Patent application title: TRANSGENIC NON-HUMAN MAMMAL FOR PRODUCING CHIMERIC HUMAN IMMUNOGLOBULIN E ANTIBODIES
Inventors:
Michel Cogne (Isles, FR)
Brice Laffleur (Limoges, FR)
Armelle Cuvillier (Saint-Jouvent, FR)
Marie Bosselut (Condat-Sur-Vienne, FR)
Assignees:
B Cell Design
UNIVERSITE DE LIMOGES
IPC8 Class: AA01K67027FI
USPC Class:
800 6
Class name: Method of using a transgenic nonhuman animal to manufacture a protein which is then to be isolated or extracted the protein is isolated or extracted from blood or serum the protein is an immunoglobulin
Publication date: 2016-06-09
Patent application number: 20160157468
Abstract:
The invention relates to a transgenic non-human mammal comprising human
immunoglobulin mu and epsilon heavy-chain constant transgenes C.mu. and
C.epsilon. inserted in place of endogenous mu heavy-chain switch sequence
S.mu., and its use for producing chimeric human immunoglobulin E
antibodies specific for an antigen of interest.Claims:
1. A transgenic non-human mammal, which comprises an endogenous
immunoglobulin heavy-chain locus comprising the replacement of its mu
heavy-chain switch sequence S.mu. by a human transgenic DNA construct
comprising respectively from its 5' to its 3'end, at least: (a) a first
site-specific recombination sequence, a human immunoglobulin mu
heavy-chain constant gene C.mu. or a functional fragment thereof
comprising at least human CH1, CH2, CH3 and CH4 exons and human membrane
M1 and M2 exons, and a second site-specific recombination sequence, said
first and said second recombination sequences being in the same
orientation and capable of site-specific recombination, and (b) a human
immunoglobulin epsilon heavy-chain constant gene C.epsilon. or a
functional fragment thereof comprising at least human CH1, CH2, CH3 and
CH4 exons and human membrane M1 and M2 exons, and wherein said transgenic
mammal comprises endogenous B-cells which produce chimeric human
immunoglobulin M (IgM) class antibodies and no endogenous IgM antibodies
from said transgenic mammal, and wherein said B-cells switch chimeric
human antibodies class production from immunoglobulin M to immunoglobulin
E (IgE) following site-specific recombination between said first and
second site-specific recombination sequences.
2. The transgenic mammal according to claim 1, wherein said site-specific recombination sequences are loxP sites of Cre recombinase.
3. The transgenic mammal according to claim 1, wherein said human transgenic DNA construct comprises the sequence SEQ ID NO: 6.
4. The transgenic mammal according to claim 1, which further comprises one or more of a human immunoglobulin light chain transgene, a human or humanized high affinity IgE receptor transgene and a transgene encoding a recombinase specific for said recombination sequences.
5. The transgenic mammal according to claim 1, wherein the transgenic mammal is a transgenic mouse.
6. A method for producing a chimeric human IgM antibody specific for an antigen of interest, comprising at least: (a) contacting a transgenic non-human mammal according to claim 1 with the antigen of interest to induce IgM antibody production in the B-cells of said mammal, and (b) collecting said IgM antibody.
7. A method for producing a chimeric human IgE antibody specific for an antigen of interest, comprising at least: (a) contacting a transgenic non-human mammal according to claim 1 with the antigen of interest to induce IgM antibody production in the B-cells of said mammal, (b) inducing site-specific recombination between said first and second recombination sequences of the transgenic DNA construct from the B-cells of said mammal using a recombinase specific for said recombination sequences to elicit IgE production in said B-cells, and) (c) collecting said IgE antibody.
8. The method according to claim 7, wherein said site-specific recombination is induced in vitro in B-cells harvested from said transgenic mammal and further immortalized.
9. The method of claim 8, wherein the site-specific recombination is induced by introducing into the immortalized B-cells an expression vector encoding said recombinase, said recombinase, or an inductor of said recombinase.
10. The method according to claim 6, wherein the antibody is collected from B-cells which that have been harvested from said mammal and further immortalized.
11. A non-human mammal embryonic stem cell comprising the human transgenic DNA construct as defined in claim 1 inserted in its endogenous immunoglobulin heavy-chain locus in place of Su.
12. A B-cell harvested from a non-human transgenic mammal according to claim 1, wherein the B-cell has been immunized with an antigen of interest, wherein said B-cell produces a chimeric human IgM or IgE antibody specific for said antigen of interest, and wherein said B-cell has been immortalized.
13. The B-cell according to claim 12, wherein the B-cell is an IgE producing B-cell further comprising a site-specific recombinase-induced deletion of its human immunoglobulin mu heavy-chain constant transgene C.mu..
14. A kit for producing chimeric human IgM or IgE antibodies specific for an antigen of interest, comprising at least: a non-human transgenic mammal according to claim 1, or a B-cell harvested from the non-human transgenic mammal according to claim 1, wherein the B-cell has been immunized with an antigen of interest, wherein said B-cell produces a chimeric human IgM or IgE antibody specific for said antigen of interest, and wherein said B-cell has been immortalized.
Description:
[0001] The present invention relates to a transgenic non-human mammal
comprising human immunoglobulin mu and epsilon heavy-chain constant
transgenes C.mu. and C.epsilon. inserted in place of endogenous mu
heavy-chain switch sequence S.mu., and its use for producing chimeric
human immunoglobulin E antibodies specific for an antigen of interest.
[0002] Human immunoglobulins E (IgE) constitute the most potent class of antibody for triggering very strong immune responses even when present at very low concentrations. IgE also constitute rare reagents only present in minute amounts in human serum and which are of interest for the diagnosis of allergy. However, because they are poorly available they were also rarely studied as potential drug for human therapy, although few reports have documented their efficacy against several targets, including tumor cells (Karagiannis et al., J. Immunol., 2007, 179, 2832-2843; Teo et al., Cancer Immunol. Immunother., 2012, 61, 2295-2309).
[0003] Like serum IgE, B cells that express IgE (IgE.sup.+ B cells) are so rare, transient and and short-lived in vivo in both human and animals that they are extremely difficult if not almost impossible to detect, identify and extract for studies. For these reasons, the knowledge about B cells that undergo class switch recombination to IgE, their terminal differentiation into IgE-secreting plasma cells and the nature of IgE memory, if any, is very limited and it has never been possible until now to design efficient systems for the production of a large repertoire of human, humanized or chimeric human IgE antibodies.
[0004] The isolation of human IgE (hIgE) from patients yields very small amounts of hIgE, and only for a limited type of antigens. In addition, the hIgE produced by said method are potentially hazardous due to possible contamination with human pathogens.
[0005] The production of recombinant human IgE comprises cloning the variable region from the heavy chain of a mouse monoclonal antibody specific for an antigen of interest and rearranging it with a human immunoglobulin epsilon heavy chain constant gene Cepsilon. This method is long, costly and produces only one human IgE antibody molecule specific for a particular antigen of interest so that the method needs to be reiterated entirely for each new human IgE antibody molecule specific for a particular antigen of interest.
[0006] Humanized transgenic mouse strains comprising from large fragments to complete human immunoglobulin heavy chain (IgH) and kappa light chain (Ig.kappa.) loci and inactivated mouse IgH and Ig.kappa. loci have been generated and successfully used for generating high-affinity antibodies against various targets (XenoMouse.RTM.: Green et al., Nature Genetics, 1994, 7, 13-21; International PCT Application WO 94/02602; Mendez et al., Nature Genetics, 1997, 15, 146-156; Hu-MAb-Mouse.RTM.: Lonberg et al., Nature, 1994, 368, 856-859; International PCT Application WO 92/03918; KM MouseTM; Tomizuka et al., Proc. Natl. Acad. Sci. USA, 2004, 97, 722-727; International PCT Application WO 02/43478). However, the frequency of IgE.sup.+ B cells in these transgenic mice which is as low as in humans does not allow the production of high amounts of human IgE antibodies.
[0007] Transgenic mice in which IgM expression is constitutively turned into IgA or IgG expression have been generated and successfully used for producing chimeric human IgG and IgA antibodies specific for various antigens of interest (International PCT Applications WO 2005/047333 and WO 2009/106773; Duchez et al., Proc. Natl. Acad. Sci. USA, 2010, 107, 3064-3069). However, IgE.sup.+ B-cells are so transient and short-lived that it is not possible to construct similar transgenic mice to produce human chimeric IgE antibodies.
[0008] The inventors have made a technological breakthrough by designing the first transgenic animals that can be used for generating hybridomas producing high amounts of high affinity chimeric human IgE antibodies of defined specificity, very easily and quickly.
[0009] Therefore, the invention relates to a transgenic non-human mammal, which comprises an endogenous immunoglobulin heavy-chain (IgH) locus comprising the replacement of its mu heavy-chain switch sequence S.mu. by a human transgenic DNA construct comprising respectively from its 5' to its 3' end, at least:
[0010] (a) a first site-specific recombination sequence, a human immunoglobulin mu heavy-chain constant gene C.mu., or a functional fragment thereof, and a second site-specific recombination sequence, said first and said second recombination sequences being in the same orientation and capable of site-specific recombination, and
[0011] (b) a human immunoglobulin epsilon heavy-chain constant gene C.epsilon. or a functional fragment thereof, and wherein said transgenic mammal comprises endogenous B-cells which produce chimeric human immunoglobulin M (IgM) class antibodies and no endogenous IgM antibodies from said transgenic mammal, and wherein said B-cells switch chimeric human antibodies class production from immunoglobulin M (IgM) to immunoglobulin E (IgE), following site-specific recombination between said first and second site-specific recombination sequences.
[0012] In the present invention:
[0013] a "site-specific recombination sequence" refers to a recombination site for a site-specific recombinase.
[0014] a "chimeric human antibody" and a "humanized antibody", both refer to an antibody comprising heavy chains with a human constant region and a variable region from said transgenic mammal and light chains in which each of the variable and constant region is human or from said transgenic mammal.
[0015] a "functional fragment of a human heavy-chain constant region gene" refers to fragment of said gene which comprises at least human CH1, CH2, CH3 and CH4 exons and human membrane M1 and M2 exons.
[0016] In the transgenic mammal of the invention, the human heavy-chain constant genes C.mu. (C mu or Cmu) and C.epsilon. (C epsilon or Cepsilon) which are inserted in place of the switch sequence S.mu. (S mu or Smu), are therefore located between the intronic activator E.mu. (E mu or Emu) and the endogenous mouse C.mu. gene (FIG. 1). The S.mu. sequence which is deleted in said transgenic mammal comprises at least positions 136442 to positions 140100 (SEQ ID NO: 1) with reference to mouse chromosome 12 sequence Genbank/EMBL accession number AC073553.
[0017] The human Cmu transgene in its original configuration (before site-specific recombination has occurred) and the human Cepsilon transgene, after site-specific recombination has occurred, are operably linked to the endogenous non-human mammal, for example mouse, heavy-chain variable region genes (V, D, J segment genes) and regulatory sequences (promoter, enhancer(s)).
[0018] In this transgenic mammals, named as Mu-Epsilon or InEps, the deletion of the endogenous mu heavy-chain switch sequence Smu associated with the insertion of the human Cmu and Cepsilon transgenes in place of this sequence, abolishes the expression of the endogenous C.mu. gene responsible for the synthesis of endogenous IgM heavy-chains and reduces drastically the expression of the other endogenous immunoglobulin heavy-chain genes, by disrupting the phenomenon of class switch recombination which is dependent on the Smu region to occur and to change to the class of the expressed immunoglobulin isotype.
[0019] The transgenic mammals of the invention produce chimeric human IgMs whose heavy chains comprise a human IgM constant region and a variable region from said transgenic mammal, and no endogenous immunoglobulins M.
[0020] The chimeric human antibodies generated by the transgenic mammal, in response to an antigen, benefit from a completely diversified repertoire since their heavy-chains whose variable domain VH mainly contribute to the formation of the antibody site correspond to the normal repertoire generated by the rearrangements of the VH, D and JH segments of the non-human mammal, for example murine, IgH locus.
[0021] Following immunization with an antigen of interest, the transgenic mammals of the invention produce high-affinity human-IgM positive (hIgM.sup.+) B cells producing high affinity chimeric human IgMs specific for said antigen.
[0022] In addition, following induction of site-specific recombination in vitro or in vivo, the B-cells of the non-human transgenic mammals switch expression of chimeric human antibodies isotype from IgM to IgE, to produce functional human-IgE positive (hIgE.sup.+) B cells expressing high affinity chimeric human IgE antibodies specific for said antigen.
[0023] Therefore, the non-human transgenic mammal according to the invention has the advantage of generating hybridomas producing high-affinity chimeric human monoclonal IgM or IgE antibodies, specific for any antigen of interest, at levels similar to those usually observed with hybridomas. The methods for producing humanized IgE antibodies specific for an antigen of interest that use the transgenic mammal of the invention are thus much more simple, efficient, rapid and economical than the methods of the prior art.
[0024] In addition, this non-human transgenic mammal which produces detectable levels of functional IgE.sup.+ B cells can also be used as model to study IgE.sup.+ B cells maturation in vivo or in vitro following site-specific recombinase induced IgM to IgE switch in the non-human transgenic mammal B cells.
[0025] Furthermore, the non-human transgenic mammal which produces detectable levels of functional chimeric human IgE.sup.+ B expressing high affinity chimeric human IgE antibodies comprising human IgE heavy chain constant region can also be used as model to study IgE antibodies functions by recruiting human effector cells, for example in double transgenic non-human mammals further expressing a human or humanized IgE receptor.
[0026] According to a preferred embodiment of said non-human transgenic mammal, said human heavy-chain constant genes Cmu and Cepsilon, each comprise human CH1, CH2, CH3 and CH4 exons and human membrane M1 and M2 exons, separated by the corresponding introns. Preferably, said human heavy-chain constant gene Cmu comprises the sequence SEQ ID NO: 2 and said human heavy-chain constant gene Cepsilon comprises the sequence SEQ ID NO: 3.
[0027] According to another preferred embodiment of said non-human transgenic mammal, the site-specific recombination sequences are loxP sequences (SEQ ID NO: 5) of Cre recombinase.
[0028] According to yet another preferred embodiment of said non-human transgenic mammal, the human transgenic DNA construct further comprises a selection marker gene. The selection marker gene, which is capable of selecting mammalian cells having integrated the human transgenic DNA construct in their genome, is advantageously the neomycine resistance gene (SEQ ID NO: 4). The selection marker gene is preferably flanked by site-specific recombination sequences. More preferably, the selection marker gene is inserted between the second site-specific recombination sequence (situated immediately downstream of the end of the human Cmu gene) and a third site-specific recombination site situated immediately upstream of the start of human Cepsilon gene, wherein the three site-specific recombination sequences are in the same orientation and the third site-specific recombination sequence is capable of site-specific recombination with the first and/or the second one. The site-specific recombination sequences are advantageously loxP sequences (SEQ ID NO: 5) of Cre recombinase. The selection marker gene is used to select the homologous recombinant non-human mammal embryonic stem cells having integrated the transgenic DNA construct in the targeted endogenous IgH locus. After selection of the homologous-recombinant cells, it can be deleted, in vitro or in vivo, using a site-specific recombinase.
[0029] According to an advantageous arrangement of said embodiment, said human transgenic DNA construct comprises or consists of the sequence SEQ ID NO: 6.
[0030] According to another preferred embodiment of said non-human transgenic mammal, it is homozygous for said human transgenic DNA construct.
[0031] According to yet another preferred embodiment of said non-human transgenic mammal, it further comprises a human immunoglobulin light chain transgene, preferably a human immunoglobulin kappa light chain transgene.
[0032] According to yet another preferred embodiment of said non-human transgenic mammal, it further comprises a human or humanized high affinity IgE receptor (FccRI) transgene. Preferably, said non-human transgenic mammal is a knock-in transgenic mammal comprising the replacement of its endogenous high affinity IgE receptor alpha-chain (.alpha.-chain) gene with its human homologue. An example of this transgenic mammal is described in Dombrowicz et al., J. Immunol., 1996, 157, 1645-1651).
[0033] According to yet another preferred embodiment of said non-human transgenic mammal, it further comprises a transgene encoding a site-specific recombinase specific for said recombination sequences, preferably a transgene encoding an inducible recombinase, more preferably a Cre recombinase transgene encoding an inducible Cre, such as for example a tamoxifen-inducible Cre recombinase. An example of this transgenic mammal is described in Metzger, D. and Chambon, P., Methods, 2001, 24, 71-80.
[0034] The transgenic mammal is advantageously homozygous for said transgene(s).
[0035] According to an advantageous arrangement of the previous embodiments it is a multiple transgenic mice comprising at least one, preferably at least two, additional transgenes chosen from a human immunoglobulin light chain transgene, a human or humanized high affinity IgE receptor transgene and transgene encoding a recombinase specific for said recombination sequences, as defined above.
[0036] The transgenic non-human mammal of the invention comprises said human Cmu and Cepsilon transgenes and eventually said additional transgenes in all its somatic and germ cells. Therefore, the transgenes are transmitted to the transgenic non-human mammal progeny. The invention encompasses also the progeny of said transgenic non-human mammal.
[0037] The invention encompasses transgenic animals obtained from any mammalian species, preferably from laboratory mammals, more preferably from laboratory rodents.
[0038] According to yet another preferred embodiment of said transgenic mammal, it is a transgenic mouse.
[0039] The transgenic mammal of the invention is useful for producing chimeric human antibodies of IgM or IgE isotype, specific for an antigen of interest.
[0040] Therefore a subject of the present invention is the use of the transgenic mammal of the invention for producing chimeric human antibodies of IgM or IgE isotype, specific for an antigen of interest.
[0041] Another subject of the present invention is a method for producing a chimeric human IgM antibody specific for an antigen of interest, comprising at least:
[0042] a) contacting a transgenic non-human mammal of the invention with the antigen of interest, to induce IgM antibody production in the B-cells of said mammal, and
[0043] b) collecting said IgM antibody.
[0044] Another subject of the present invention is a method for producing a chimeric human IgE antibody specific for an antigen of interest, comprising at least:
[0045] a) contacting a transgenic non-human mammal of the invention with the antigen of interest, to induce IgM antibody production in the B-cells of said mammal,
[0046] b) inducing site-specific recombination between said first and second recombination sequences of the human transgenic DNA construct from the B-cells of said mammal, to elicit IgE production in said B-cells, and
[0047] c) collecting said IgE antibody.
[0048] The antigen which is used in the present invention is any natural, recombinant or synthetic substance which is able to induce a specific immune response, including the production of specific antibodies, when introduced into a non-human transgenic mammal according to the invention. For example, the antigen, comprises one or more of a protein, peptide, lipid, sugar, nucleic acid, and/or mineral.
[0049] Step a) of the methods is performed by immunizing the transgenic non-human mammal with the antigen of interest according to standard protocols which are known in the art.
[0050] The antibody may be collected from the serum or B-cell(s) harvested from said mammal. Preferably, the antibody is collected from B-cell(s) which have been harvested from said mammal and further immortalized.
[0051] The site-specific recombination may be induced in vivo, in the non-human transgenic mammal or in vitro, in the B-cells harvested from said transgenic mammal and, preferably, further immortalized.
[0052] The induction in vivo is preferably performed using a transgenic mammal comprising a site-specific recombinase gene encoding a recombinase specific for said recombination sequences, more preferably, said recombinase gene is an additional transgene of the transgenic mammal coding for an inducible recombinase, as defined before.
[0053] The induction in vitro is preferably performed by immortalizing the B-cells harvested from the transgenic mammal and introducing into the immortalized B cells, an expression vector which encodes the site-specific recombinase, the site-specific recombinase itself or the site-specific recombinase inductor, when the transgenic mammal comprises a recombinase transgene, as defined before.
[0054] The expression vector is advantageously an expression plasmid comprising a recombinase gene encoding a site-specific recombinase specific for said recombination sequences, wherein said recombinase gene is expressible, in vitro, in said B-cells. For example, the recombinase gene is under the control of a ubiquitous promoter, such as the CMV promoter. The site-specific recombinase which is introduced into the cells is advantageously fused to a cell penetrating peptide (CPP) sequence such as for example the sequence of a CPP derived from HIV Tat basic domain.
[0055] According to an advantageous embodiment of said method, the site-specific recombination is induced in vitro in the B-cells harvested from said transgenic mammal and further immortalized.
[0056] B-cell immortalization is performed by fusion of the B-cells with a myeloma cell line, a lymphoblastoid cell line, lymphoma cells or an heteromyeloma cell line, according to standard hybridoma production techniques. Preferably, the B-cells are immortalized by fusion with a murine myeloma cell line, more preferably a murine myeloma cell line like the SP2/0 cell line, which does not produce any murine antibody, is immortalized, and possesses the entire secretion machinery necessary for the secretion of immunoglobulins. The immortalized B-cells are screened for specific antibody production using conventional assays like ELISA. After screening, they are usually cloned using standard methods. The antibodies which are secreted by the immortalized B-cells are harvested from the extracellular medium and usually further purified by conventional techniques known to the persons skilled in the art, such as affinity chromatography.
[0057] The chimeric human IgM or IgE antibodies specific for an antigen of interest which are produced by the methods of the invention are polyclonal or monoclonal antibodies. Preferably, said antibodies are monoclonal antibodies. These antibodies, in particular the IgE antibodies, are useful for diagnosis and therapy of human diseases in particular, allergy and cancer. The chimeric human IgE antibodies specific for an antigen of interest are used as standards and controls in human allergy assays. Alternatively, they are use as therapeutic antibodies for cancer immunotherapy.
[0058] The subject of the present invention is also an isolated polynucleotide comprising the human transgenic DNA construct as defined above. The isolated polynucleotide, either synthetic or recombinant, may be DNA, RNA or combination thereof, either single- and/or double-stranded, preferably double-stranded DNA. According to another preferred embodiment, the polynucleotide further comprises DNA fragments from the endogenous IgH locus of said mammal comprising the sequences immediately upstream and downstream of its switch sequence S.mu., and said DNA fragments being inserted immediately upstream of the first site-specific recombination sequence and immediately downstream of the human Cepsilon gene, respectively of the human transgenic DNA construct as defined above. The DNA fragments, named 5'and 3' homology arms or 5' and 3' arms, are advantageously of about 5 kb. The DNA fragments correspond advantageously to mouse JH/E.mu. and C.mu. regions, respectively. Preferably, the DNA fragments are from mouse IgH locus. More preferably, the DNA fragments comprise positions 131281 to 136441 (SEQ ID NO: 7) and 140101 to 145032 (SEQ ID NO: 8), respectively of mouse chromosome 12 sequence Genbank/EMBL accession number AC073553.
[0059] According to another preferred embodiment, the polynucleotide comprises or consists of the sequence SEQ ID NO: 9.
[0060] Another subject of the present invention is a targeting vector comprising the human transgenic DNA construct flanked by 5'and 3' homology arms, as defined above. Vectors include usual vectors used in genetic engineering including for example plasmids and viral vectors. The targeting vector is useful for producing the transgenic mammal of the invention. According to a preferred embodiment, said targeting vector is a plasmid. According to another preferred embodiment, said targeting vector comprises or consists of SEQ ID NO: 10.
[0061] The subject of the present invention is also an isolated cell comprising the human transgenic DNA construct as defined above, with the exclusion of human embryonic stem cells. The cell may be eukaryotic or prokaryotic. According to a preferred embodiment, said cell is a homologous-recombinant cell comprising the human transgenic DNA construct inserted in its endogenous IgH locus, in place of Su. Said homologous-recombinant cell is advantageously obtained by introducing the targeting vector as defined above in a cell, thus allowing the insertion of the transgenic DNA construct in the targeted IgH locus.
[0062] According to another preferred embodiment, said cell is an embryonic stem cell from a non-human mammal. Preferably, the non-human mammal embryonic stem cell is a homologous-recombinant cell as defined above. The homologous recombinant stem cell is useful for producing the non-human transgenic mammal according to the invention.
[0063] According to yet another preferred embodiment, said cell is a B-cell harvested from a non-human transgenic mammal of the invention immunized with an antigen of interest, which B-cell produces a chimeric human antibody of IgM or IgE isotype, specific for an antigen of interest. According to an advantageous arrangement of said embodiment, said B-cell has been further immortalized. Said immortalized B-cells is advantageously an hybridoma. Preferably, said immortalized B-cell is an IgE producing B-cell further comprising a site-specific recombinase-induced deletion of its human Cmu transgene.
[0064] The polynucleotide, targeting vector and non-human mammal embryonic stem cell according to the invention are useful for producing the non-human transgenic mammal of the invention. The B-cell harvested from a non-human transgenic mammal of the invention immunized with an antigen of interest, in particular the immortalized B-cell is useful for producing human chimeric IgM and IgE antibodies specific for an antigen of interest.
[0065] Another subject of the present invention is a kit for producing chimeric human IgM or IgE antibodies specific for an antigen of interest, comprising at least:
[0066] a non-human transgenic mammal of the invention, and eventually, an antigen of interest, or
[0067] a B-cell harvested from a non-human transgenic mammal of the invention immunized with said antigen of interest, which B-cell produces a chimeric human antibody of IgM or IgE isotype, specific for an antigen of interest, preferably a B-cell which has been further immortalized, more preferably an IgE producing B-cell further comprising a site-specific recombinase-induced deletion of its human Cmu transgene.
[0068] The practice of the present invention will employ, unless otherwise indicated, conventional techniques which are within the skill of the art. Such techniques are explained fully in the literature.
[0069] The different sequences which are used for constructing the polynucleotide and derived targeting vector, cell, transgenic mammals of the invention, including the sequences of human and non-human mammal immunoglobulin genes, are known in the art and accessible in databases. The polynucleotide and targeting vector according to the invention are constructed and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques using classical methods, according to standard procedures as those described in: Current Protocols in Molecular Biology (Frederick M AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA) and Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). For example, the polynucleotide is produced by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, by total or partial chemical synthesis, or by a combination of said techniques. The insertion of gene fragments into the genome of non-human mammals may be carried out in a random manner, preferably it is carried out in a targeted manner, by homologous recombination with an appropriate targeting vector optionally comprising recombination sequences for a site-specific recombinase such as the loxP sites of Cre recombinase. The inactivation or deletion of gene fragments in the genome of non-human mammals is carried out by homologous recombination with an appropriate targeting vector optionally comprising recombination sequences for a site-specific recombinase such as the loxP sites of Cre recombinase. The transgenic animals according to the invention are obtained by conventional methods for animal transgenesis, according to the standard protocols as described in Transgenic Mouse: Methods and Protocols; Methods in Molecular Biology, Clifton, N.J., Volume 209, October 2002, edited by: Marten H Hofker, Jan Van Deursen, Marten H Hofker and Jan Van Deursen, published by Holly T Sklar: Humana Press. The multiple transgenic animals are obtained by crossing animals transgenic for human mu-epsilon heavy-chains with one or more animals transgenic for human immunoglobulin light chain, human or humanized high affinity IgE receptor, and/or a site-specific recombinase, as defined above. The chimeric human IgM and IgE antibodies are prepared by conventional techniques known to persons skilled in the art, such as those described in Antibodies: A Laboratory Manual, E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988.
[0070] In addition to the preceding features, the invention also comprises other features which will emerge from the description which follows, which refers to examples of production and use of transgenic non-human mammals according to the present invention and to the appended drawings in which:
[0071] FIG. 1 represents the construction of transgenic mice by knock-in of a human C mu/neo/human C epsilon cassette downstream of the endogenous mouse JH region. wt mouse IgH locus is represented (not scale) with the end of the VH(D)JH cluster, the switch mu region and the first endogenous mouse mu and delta heavy chain constant genes. The targeting vector contains: the 3'arm corresponding to the mouse DJH cluster, a floxed human mu heavy chain constant gene, a floxed Neo resistance gene, a human epsilon heavy chain constant gene and the 3'arm corresponding to the mouse endogenous mu heavy chain constant gene. After homologous recombination in ES cells the S mu region is replaced by the human genes. Human IgM (hIgM) is expressed and allows B cell development and hIgM production. After deletion of the floxed genes upon Cre-loxP-mediated recombination (named herein as creting) in vitro or in vivo, hIgE production occurs in vitro, in hybridomas or in vivo, in B-cells.
[0072] FIG. 2 represents the sequence of the targeting vector (SEQ ID NO: 10) used for constructing the Mu-Epsilon (also named as InEps) transgenic mice. Exons are in bold. loxP sequences are highlighted in grey. PolyA signals are boxed. Core Emu sequence is underlined. Vector sequences are in italics.
[0073] FIG. 3 illustrates the validation of the construction at the genomic and protein level. (A) Screening of ES cells after transfection of the knock-in vector. Genomic DNA was digested with EcoRI and a mouse C delta probe was used to detect recombinant clones. Wt allele results in a band of about 8 kb and recombinant allele gives a band of about 4 kb. (B) Screening of human IgM expression on blood lymphocytes. Lymphocytes were stained with anti-mouse CD19 FITC (eB1D3; BECKMAN COULTER), anti-mouse IgM PE (eB121-15F9; BECKMAN COULTER) and anti-human IgM APC (SA-DA4; BECKMAN COULTER) antibodies. B cells were gated on CD 19+ lymphocytes and analysed for human IgM expression. Homozygote mice, named as Mu-Epsilon or InEps mice, expressed only human IgM. These facs are representatives of at least ten mice per groups.
[0074] FIG. 4 illustrates in vitro creting of hybridomas and splenocytes and hIgE production. (A) Hybridomas from InEps mice were selected for hIgM production (left panel) and stained intracellularly with anti-human IgM APC (SA-DA4) and anti-human IgE FITC (A80-108F; BECKMAN COULTER). After deletion of the human mu heavy chain constant gene, human IgE expression was detected in the three independent tests (right panels). (B) Splenocytes from wt mice, InEps mice and InEps.times.CreTamox mice were cultured two days in vitro in presence of LPS (10 .mu.g/mL) and 4-hydroxytamoxifen (500 nM, SIGMA H7904) in order to activate Cre recombinase activity into InEps.times.CreTamox mice. Cells were stained in intracellular with anti-mouse CD19 APC-H7 (1D3; BECKMAN COULTER), anti-human IgM APC (SA-DA4) and anti-human IgE FITC (A80-108F). Wt cells do not express hIgM or hIgE (left panel), InEps mice only express hIgM (middle panel). hIgE expression was only detected in IpEps.times.CreTamox mice in the three independent tests (right panels).
[0075] FIG. 5 illustrates in vivo creting of B-cells and chimeric human IgE production in InEps.times.CreTamox double transgenic mice. (A and B). Intracellular staining of total hIgE+ cells from spleen of InEps (.mu..epsilon.KI) and InEps.times.CreTamox.times.CreTamox, also named .mu..epsilon.KI.times.CreERT2) transgenic mice. (C and D). BrdU incorporation in hIgE+ and hIgM+ cells from spleen and bone marrow of InEps.times.CreTamox transgenic mice. (E). Kinetic of hIgE production in sera of InEps (.mu..epsilon..kappa.I) and InEps.times.CreTamox (.mu..epsilon..kappa.I.times.CreTamox) transgenic mice.
[0076] FIG. 6 illustrates specific polyclonal hIgM response anti-antigen in InEps transgenic mice InEps transgenic mice were immunized by intraperitoneal route with a specific protein antigen from a human pathogen (50 .mu.g/immunization/mouse) (in ratio 1:1 with Freund Adjuvant) four times every two weeks. Sera were sampled before immunization (DO) and 2 weeks after each immunization and stored at -20.degree. C. until the ELISA was performed.
[0077] FIG. 7 illustrates anti-Ag affinity of Mab IgM clone and Mab IgE-switched clone. Specific anti-antigen IgM expressing clones were transfected with Cre recombinase expression plasmid to excise the human mu-IgH gene and allow the splicing between the rearranged variable genes and the human epsilon-IgH gene. After 5 days of culture, the culture supernatants of the isolated clones were tested by anti-human IgE ELISA.
[0078] FIG. 8 illustrates monoclonal chimeric human IgM and IgE production in hybridomas derived from Mu-Epsilon transgenic mice. Proteins were analyzed by western-blot using (A) mouse anti-human IgM (SADA4 clone; BECKMAN COULTER) or (B) mouse anti-human IgE (Depsilon 2; BECKMAN COULTER). Lane 1: supernatant of Hybridoma. Lane:4 supernatant of the same mab IgM.sup.+ clone after in vitro Cre-induced IgM to IgE switch. Lane 2 & 3: Protein MW ladder (Bio-Rad) in kDalton Arrows in A shows monoclonal chimeric human IgM and arrows in B shows monoclonal chimeric human IgE.
EXAMPLE 1
Production and Characterization of Mu-Epsilon Transgenic Mice
1) Construction of the Homologous Recombination Targeting Vector
[0079] DNA manipulations were performed using standards protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc, Library of Congress, USA).
[0080] Human mu and epsilon heavy chain constant genes were amplified from human genomic DNA with specific primers containing restriction sites and subcloned into TopoTA 2.1 vectors (INVITROGEN) to yield Topo-hIgM and Topo-hIgM vectors. The vector containing the two arms and the Neo resistance gene (V588) has been described previously (Duchez et al., Proc. Natl. Acad. Sci. U.S.A., 2010, 107: 3064-3069 and WO 2005/047333). Human epsilon heavy chain constant gene was ligated after XhoI restriction of the Topo-hIgE vector and SalI restriction of the V588 vector. A loxP site has been introduced in 5' of the human mu heavy chain constant gene resulting in a ClaI-ClaI fragment, cloned into the ClaI unique site of the V588+hIgE vector (FIG. 1). The resulting targeting vector comprising the human mu and epsilon heavy chain constant genes (human mu-IgH and epsilon-IgH genes; FIG. 1) has the sequence SEQ ID NO: 10 (FIG. 2). The human transgenic construct has the sequence SEQ ID NO: 9.
2) Transfection of ES Cells and Injection into Blastocysts
[0081] After sequencing, the targeting vector has been linearized with PvuI and transfected into E14 ES cells with Amaxa.RTM. technology according to the manufactor's instructions. Neomycin (300 .mu.g/mL) was added 24 h later to select ES cells. Resistant clones were screened by Southern blot after EcoRI restriction using a mouse Cdelta probe (Laffleur et al., Methods Mol. Biol. 2012, 901, 149-159). Wild type allele results in a fragment of about 8 kb and recombinant allele in a band of about 4 kb (FIG. 3A). One clone (114) was used for micro-injection and three agouti positive mice were obtained.
3) Screening of Mu-Epsilon (or InEps) Transgenic Mice by Flow Cytometry
[0082] Mice were screened on blood by flow cytometry with a three colors staining against mouse CD19 (FITC eB1D3), mouse IgM (PE eB121-15F9) and human IgM (APC SA-DA4) clone. Cells were analyzed on a Becton Dickinson Fortessa LSRII. B cells were gated on CD19+ lymphocytes and analysed for human IgM expression. Homozygote mice, named hereafter as Mu-Epsilon or InEps mice, expressed only human IgM (FIG. 3B).
4) In Vitro Creting of Hybridomas and Splenocytes and Chimeric Human IgE Production
[0083] 4.1) hIgE Production from InEps Hybridomas Transiently Expressing Cre Recombinase
[0084] Hybridomas were generated from InEps mice according to standard techniques (Kohler G, Milstein C, Nature, 1975, 256, 495-497) and selected for hIgM production (FIG. 4A, left panel) 10.sup.6 hIgM.sup.+ hybridoma cells were incubated with 2 .mu.g of a pCDNA3/Cre-recombinase expression vector complexed with 24 of 293fectin.TM. (INVITROGEN) for 4 h in Opti-MEM.RTM. medium (INVITROGEN) at 37.degree. C. in CO.sub.2 atmosphere. The cells were spread over five culture plates (96-well plates, NUNC) in culture medium (DMEM high Glucose/Glutamax/10% Calf Bovine Serum). After 5 days of culture, the hybridoma cells were stained with anti-human IgM APC (SA-DA4) and anti-human IgE FITC (A80-108F).
[0085] Transient expression of Cre recombinase enzyme excises the human mu-IgH transgene and allows the splicing between the rearranged variable genes from the mouse and the human epsilon-IgH transgene, resulting in chimeric human IgE expression (FIG. 4A, right panels).
4.2) hIgE Production from InEps.times.CreTamox Splenocytes Comprising a Tamixofen-Dependent Cre Recombinase
[0086] InEps mice were bred with CMV-Cre-ER.sup.T transgenic mice (Metzger D. and Chambon P., Methods, 2001, 24, 71-80), a transgenic strain expressing a Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (ER), under the control of the ubiquitous cytomegalovirus major IE gene enhancer/promoter, resulting in a tamoxifen-dependent Cre recombinase which is active in a number of cell types, including B cells compartments. The double-transgenic mouse strain homozygous for human Mu-Epsilon IgH genes and at least heterozygous for the CMV-Cre-ER.sup.T transgene is called InEps.times.CreTamox, .mu..epsilon.KI.times.CreTamox, or .mu..epsilon.KI.times.CreERT2.
[0087] Splenocytes were isolated from wild-type, InEps and InEps.times.CreTamox mice according to conventional methods and cultured two days in vitro in presence of LPS (10 .mu.g/mL) and 4-hydroxytamoxifen (500 nM, SIGMA H7904) in order to activate Cre recombinase activity into InEps.times.CreTamox mice. Cells were stained with anti-mouse CD19 APC-H7 (1D3), anti-human IgM APC (SA-DA4) and anti-human IgE FITC (A80-108F).
[0088] Wt cells do not express hIgM or hIgE (FIG. 4B, left panel), InEps mice only express hIgM (FIG. 4B, middle panel). hIgE expression was only detected in IpEps.times.CreTamox mice in the three independent tests (FIG. 4B, right panels).
5) In Vivo Creting of B-Cells and Chimeric Human IgE Production
[0089] InEps and InEps Cre Tamoxifen (InEps.times.CreTamox) mice (8 to 10 weeks old) were injected intraperitonally (i.p.) with 2 .mu.g of tamoxifen (SIGMA; T5648) at day 0 and fed orally twice with a feeding needle with 8 mg tamoxifen incorporated to 500 .mu.L of water at day 1 and 2. Serum samples were collected at the indicated times (FIG. 5E) and in parallel mice were sacrified for FACS analysis. Prior to all IgE staining experiments, cells were first washed for 1 minute with 100 .mu.L of acidic buffer per million cells (Ice-cold acid buffer: 0.01 M EDTA, 0.005 M KCl, 0.085 M NaCl, 0.05 M NaAcetate (NaAc), pH=4) to remove passively bound Ig from Fc receptors, then neutralized in PBS and centrifuged.
[0090] Intracellular staining of total hIgE+ cells in spleen were performed on 10.sup.6 splenocytes with IntraPrep Permeabilization Reagent (BECKMAN COULTER; A07803) and quantified by FACS analysis (FIGS. 5A and 5B, n=2 to 5 mice per group). This experiments show the efficiency of the strategy but also the rapid decrease of IgE+ cells.
[0091] A cohort of mice were also injected intra-peritoneally (i.p.) with 200 .mu.g of bromodeoxyuridine (BrdU, Sigma B5002) into PBS at day 20 and further fed with BrdU during the time of the experiment (10 days) into water (500 .mu.g/mL). For the staining 210.sup.6 cells were washed, blocked, fixed and finally permeabilized using the BrdU Flow kit (Beckton Dickinson). BrdU epitopes were released by DNAse treatment 1 hour at 37.degree. C. Finally, intra-cellular class-specific Ig staining (IgM and IgE) was for 20 minutes at RT and BrdU staining for another 20 minutes at room temperature. BrdU incorporation was evaluated by FACS analysis (FIG. 5C and 5D, n=5 mice per group). This experiments confirm the short-fate of IgE+ cells because these cells were generated during the window of BrdU treatment and failed to accumulate as "long-lived" cell contrary to IgM+ cells.
[0092] Secreted human IgE were revealed using an anti-human IgE galactosidase and the Methyl-umbelliferyl-.beta. D galactoside as substrate. Results were expressed versus a human IgE standard curve. Mouse sera were assayed at 1:10 dilution for mouse Ig and for human IgE determinations. Kinetic of hIgE production in vivo was determined (FIG. 5E, n=2 to 5 mice per group) and shows a strong hIgE production, culminating at 15 000 kU/L (36 000 .mu.g/L) (human mean at 100 kU/mL (240 .mu.g/L)), consistent with high secretion by plasma cells.
EXAMPLE 2
Production of Antigen-Specific Chimeric Human IgE Using InEps Transgenic Mice
[0093] InEps transgenic mice were immunized by intraperitoneal route with a specific protein antigen from a human pathogen (50 .mu.g/immunization/mouse) (in ratio 1:1 with Freund Adjuvant) four times every two weeks. Sera were sampled before immunization and 2 weeks after each immunization and stored at -20.degree. C. until the ELISA was performed. Briefly, 96 well plates were coated overnight at 4.degree. C. with antigen (1 .mu.g/mL). Plates were blocked with PBS/BSA 2%. Sera were diluted 100 times in PBS/BSA 0.2% and incubated 2h at 37.degree. C. AP-conjugated secondary Ab (goat anti-human IgM, Beckman Coulter) were used at 1 .mu.g/mL to detect bound hIgM.
[0094] FIG. 6 shows that transgenic mice immunized with the antigen produce specific chimeric human IgM.
[0095] Hybridomas producing specific chimeric human monoclonal IgM were generated from the immunized transgenic mice according to standard techniques (Kohler G, Milstein C, Nature, 1975, 256, 495-497) and selected for hIgM production by ELISA (Table I).
TABLE-US-00001 TABLE I Validation of human chimeric monoclonal IgM for Diagnostic purpose, as calibrator and positive control First Second Clone validated as OD screening screening calibrator and (B-cell Screened positive positive Stabilized positive control in Harvest Collected hybridoma clone clone positive diagnostic test on day) splenocytes clone (by ELISA) (by ELISA) clone automate Mouse 1.7895 46.10.sup.6 cells 1530 clones 5 hybridoma 9 clones 5 -- #1 clones Mouse 1.721 67.10.sup.6 cells 1150 clones 9 hybridoma 40 clones 2 1 clone #2 clones Mouse 0.6295 23.10.sup.6 cells 960 clones 17 -- 3 2 clones #3 hybridoma clones
[0096] After selection of the specific anti-antigen human chimeric IgM expressing clone, transient expression of the Cre recombinase enzyme excises the human mu-IgH gene and allows the splicing between the rearranged variable genes and the human epsilon-IgH gene. Briefly, 10.sup.6 cells were incubated with 2 .mu.g of a pcDNA3/Cre-recombinase expression vector complexed with 2 .mu.L of 293fectin.TM. (for 4 h in Opti-MEM.RTM. medium at 37.degree. C. in CO2 atmosphere. The cells were spread over five culture plates (96-well plates, NUNC) in culture medium (DMEM high Glucose/Glutamax/10% Calf Bovine Serum). After 5 days of culture, the culture supernatants of the isolated clones were tested by anti-human IgE ELISA.
[0097] The affinity of transformed clones for the antigen is verified by the anti-antigen ELISA (FIG. 7).
[0098] Clones expressing anti-Ag specific human chimeric IgE were identified, subcloned then amplified in culture for IgE production. The clones produce from 0.1 to 50 .mu.g/ml of anti-Ag human chimeric IgE in standard culture conditions (T75 flask (75 cm2); DMEM high glucose, 10% FCS).
[0099] Monoclonal chimeric human IgM and IgE production was analyzed by Western-blot (FIG. 8). Proteins were separated on SDS-PAGE gels (4%-15% gradient acrylamide gels, BioRad) under non-reducing conditions and transferred on nitrocellulose membranes. The membranes were blocked with 5% milk in PBS and probed in 3% milk/PBS buffer with the primary mouse anti-human IgM (SADA4 clone, BECKMAN COULTER, 1/2000 diluted) or the primary mouse anti-human IgE (Depsilon 2, BECKMAN COULTER, 1/5000 diluted), revealed with the HRP-conjugated goat anti-mouse IgG1 (BECKMAN COULTER, 1/5000 diluted) and visualized by Peroxidase substrat on colorimetric enzymatic reaction (DAB, SIGMA).
[0100] FIG. 8 shows that the hybridomas produce chimeric human IgM before induction of site-specific recombination and human IgE after induction of site-specific recombination.
Sequence CWU
1
1
1013665DNAartificial sequencesynthetic polynucleotide (mouse Smu deletion)
1taaaagaaaa atgttgcctg ttaaccaata atcatagagc tcatggtact ttgaggaaat
60cttagaaagc gtgtatacaa ttgtctggaa ttatttcagt taagtgtatt agttgaggta
120ctgatgctgt ctctacttca gttatacatg tgggtttgaa ttttgaatct attctggctc
180ttcttaagca gaaaatttag ataaaatgga tacctcagtg gtttttaatg gtgggtttaa
240tatagaagga atttaaattg gaagctaatt tagaatcagt aaggagggac ccaggctaag
300aaggcaatcc tgggattctg gaagaaaaga tgtttttagt ttttatagaa aacactacta
360cattcttgat ctacaactca atgtggttta atgaatttga agttgccagt aaatgtactt
420cctggttgtt aaagaatggt atcaaaggac agtgcttaga tccgaggtga gtgtgagagg
480acaggggctg gggtatggat acgcagaagg aaggccacag ctgtacagaa ttgagaaaga
540atagagacct gcagttgagg ccagcaggtc ggctggacta actctccagc cacagtaatg
600acccagacag agaaagccag actcataaag cttgctgagc aaaattaagg gaacaaggtt
660gagagcccta gtaagcgagg ctctaaaaag cacagctgag ctgagatggg tgggcttctc
720tgagtgcttc taaaatgcgc taaactgagg tgattactct gaggtaagca aagctgggct
780tgagccaaaa tgaagtagac tgtaatgaac tggaatgagc tgggccgcta agctaaacta
840ggctggctta accgagatga gccaaactgg aatgaacttc attaatctag gttgaataga
900gctaaactct actgcctaca ctggactgtt ctgagctgag atgagctggg gtgagctcag
960ctatgctacg ctgtgttggg gtgagctgat ctgaaatgag atactctgga gtagctgaga
1020tggggtgaga tggggtgagc tgagctgggc tgagctagac tgagctgagc tagggtgagc
1080tgagctgggt gagctgagct aagctggggt gagctgagct gagcttggct gagctagggt
1140gagctgggct gagctggggt gagctgagct gagctggggt aagctgggat gagctggggt
1200gagctgagct gagctggagt gagctgagct gggctgagct ggggtgagct gggctgagct
1260gggctgagct gggctgagct ggggtgagct gagctggggt gagctgagct gagctggggt
1320gagctgagct gagctggggt gagctggggt gagctgagct ggggtgagct gagctgagct
1380ggggtgagct gagctggggt gagctgagct gagctggggt gagctgagct gagctgagct
1440gagctgagct ggggtgagct gagctgagct gagctggggt gagctggggt gagctgagct
1500gagctggagt gagctgagct gggctgagct ggggtgagct gggctgagct ggggtgagct
1560gagctgagct gagctgagct ggggtgagct gagctgagct ggggtgagct gagctggggt
1620gagctgggct gagctgagct gagctgagct gagctgagct gagctgagct gagctgagct
1680gagctgagct gagctgagct gagctgagct gagctggggt gagctgagct gagctgggct
1740crgagctggg gtgagctggg ctgagctggg ctgagctggg ctgagctggg gtgagctgag
1800ctsmggggtg agctgagctg agctgggctg agctgagctg agctggggtg agctgagctg
1860agctggggtg agctgagctg agctgagctg gggtgagctg agctgggctg agcagggctg
1920agctggggtg agctgagctg agctggggtg agctgggctg agctgggctg agctgagctg
1980agctgggctg agctgggctg agctgggctg agctgggctg agctgggctg agctggggtg
2040agctgagctg agctggggtg agctggggtg agctgagctg gggtgagctg agctggggtg
2100agctgagctg agctggggtg agctgagctg gggtgagctg agctgagctg gggtgagctg
2160agctgagctg gggtgagctg agctagggtg aactgggctg ggtgagctgg agtgagctga
2220gctgaggtga actggggtga gccgggatgt tttgagttga gctggggtaa gatgagctga
2280actggggtaa actgggatga gctgtggtga gcggagctgg attgaactga gctgtgtgag
2340ctgagctggg gtcagctgag caagagtgag tagagctggc tggccagaac cagaatcaat
2400taggctaagt gagccagatt gtgctgggat cagctgtact cagatgagct gggatgaggt
2460aggctgggat gagctgggct agctgacatg gattatgtga ggctgagcta gcatgggctg
2520gcctagctga tgagctaagc ttgaatgagc ggggctgagc tggactcaga tgtgctagac
2580tgagctgtac tggatgatct ggtgtagggt gatctggact caactgggct ggctgatggg
2640atgcgccagg ttgaactagg ctcagataag ttaggctgag tagggcctgg ttgagatggt
2700tcgggatgag ctgggaaaag atggactcgg accatgaact gggctgagct gggttgggag
2760accatgaatt gagctgaact gagtgcagct gggataaact gggttgagct aagaatagac
2820tacctgaatt gtgccaaact cggctgggat caattggaaa ttatcaggat ttagatgagc
2880cggactaaac tatgctgagc tggactggtt ggatgtgttg aactggcctg ctgctgggct
2940ggcatagctg agttgaactt aaatgaggaa ggctgagcaa ggctagcctg cttgcataga
3000gctgaacttt agcctagcct gagctggacc agcctgagct gagtaggtct aaactgagtt
3060aaaaatcaac agggataatt taacagctaa tttaacaagc ctgaggtctg agattgaatg
3120agcagagctg ggatgaactg aatgagtttc accaggcctg gaccagttag gctaggacct
3180cgttctatag aggcagactg tgtgctacag tggagtttca agatgattcc atgagtcctc
3240cccgccccca acataaccca ccttcctcct accctacacg cctgtctggt gtgtaaatcc
3300cagctttgtg tgctgataca gaagcctgag cccctccccc acctccacct acctattact
3360ttgggatgag aatagttctc ccagccagtg tctcagaggg aagccaagca ggacaggccc
3420aaggctactt gagaagccag gatctaggcc tctccctgag aacgggtgtt catgccccta
3480gagttggctg aagggccaga tccacctact ctagaggcat ctctccctgt ctgtgaaggc
3540ttccaaagtc acgttcctgt ggctagaagg cagctccata gccctgctgc agtttcgtcc
3600tgtataccag gttcacctac taccatatct agccctgcct gccttaagag tagcaacaag
3660gaaat
366524492DNAartificial sequencesynthetic polynucleotide (human Cmu gene)
2tcagggagtg catccgcccc aacccttttc cccctcgtct cctgtgagaa ttccccgtcg
60gatacgagca gcgtggccgt tggctgcctc gcacaggact tccttcccga ctccatcact
120ttctcctgga aatacaagaa caactctgac atcagcagca cccggggctt cccatcagtc
180ctgagagggg gcaagtacgc agccacctca caggtgctgc tgccttccaa ggacgtcatg
240cagggcacag acgaacacgt ggtgtgcaaa gtccagcacc ccaacggcaa caaagaaaag
300aacgtgcctc ttccaggtga gggccgggcc cagccaccgg gacagagagg gagccgaagg
360ggggcgggag tggcgggcac cgggctgaca cgtgtccctc actgcagtga ttgctgagct
420gcctcccaaa gtgagcgtct tcgtcccacc ccgcgacggc ttcttcggca acccccgcaa
480gtccaagctc atctgccagg ccacgggttt cagtccccgg cagattcagg tgtcctggct
540gcgcgagggg aagcaggtgg ggtctggcgt caccacggac caggtgcagg ctgaggccaa
600agagtctggg cccacgacct acaaggtgac cagcacactg accatcaaag agagcgactg
660gctcagccag agcatgttca cctgccgcgt ggatcacagg ggcctgacct tccagcagaa
720tgcgtcctcc atgtgtgtcc ccggtgagtg acctgtcccc aggggcagca cccaccgaca
780cacaggggtc cactcgggtc tggcattcgc caccccggat gcagccatct actccctgag
840ccttggcttc ccagagcggc caagggcagg ggctcgggcg gcaggacccc tgggctcggc
900agaggcagtt gctactcttt gggtgggaac catgcctccg cccacatcca cacctgcccc
960acctctgact cccttctctt gactccagat caagacacag ccatccgggt cttcgccatc
1020cccccatcct ttgccagcat cttcctcacc aagtccacca agttgacctg cctggtcaca
1080gacctgacca cctatgacag cgtgaccatc tcctggaccc gccagaatgg cgaagctgtg
1140aaaacccaca ccaacatctc cgagagccac cccaatgcca ctttcagcgc cgtgggtgag
1200gccagcatct gcgaggatga ctggaattcc ggggagaggt tcacgtgcac cgtgacccac
1260acagacctgc cctcgccact gaagcagacc atctcccggc ccaagggtag gccccactct
1320tgcccctctt ctgcactcct gcaactcctt gcctctgggg gcatggtgga aagcacccct
1380cactcccccg ttgtctgggc aactggggaa aaggggactc aaccccagcc cacaggctgg
1440tccccccact gccccgccct caccaccatc tctgttcaca ggggtggccc tgcacaggcc
1500cgatgtctac ttgctgccac cagcccggga gcagctgaac ctgcgggagt cggccaccat
1560cacgtgcctg gtgacgggct tctctcccgc ggacgtcttc gtgcagtgga tgcagagggg
1620gcagcccttg tccccggaga agtatgtgac cagcgcccca atgcctgagc cccaggcccc
1680aggccggtac ttcgcccaca gcatcctgac cgtgtccgaa gaggaatgga acacggggga
1740gacctacacc tgcgtggtgg cccatgaggc cctgcccaac agggtcaccg agaggaccgt
1800ggacaagtcc accggtaaac ccaccctgta caacgtgtcc ctggtcatgt ccgacacagc
1860tggcacctgc tactgaccct gctggcctgc ccacaggctc ggggcggctg gccgctctgt
1920gtgtgcatgc aaactaaccg tgtcaacggg gtgagatgtt gcatcttata aaattagaaa
1980taaaaagatc cattcaaaag atactggtcc tgagtgcacg atgctctggc ctactggggc
2040ggcggctgtg ctgcacccac cctgcgcctc ccctgcagaa caccttcctc cacagccccc
2100acccctgcct cacccacctg cgtgcctcag tggcttctag aaacccctga attccctgca
2160gctgctcaca gcaggctgac ctcagacttg ccattcctcc tactgcttcc agaaagaaag
2220ctgaaagcaa ggccacacgt atacaggcag cacacaggca tgtgtggata cacatggaca
2280gacacggaca cacacaaaca catggacaca cagagacgtg ctaacccatg ggcacacaca
2340tacacagaca tggacccaca cacaaacata tgtggacaca catgtacaaa catgcacagg
2400cacacaaaga gaacactgac tacaggcaca cacacacacg ggcacacaca tggatatgtg
2460cacacatgga cacatacatg tgcaggacat gcacacacac agacacacta gcacagaggc
2520atacacacac agacacacac attcacaaac acacatgtgc atgcaaacac acacacatgt
2580acagacacga gtacatggac acatgcacac ccagagacac actgacacag acacacagga
2640gcatgtgata cactaacacg tggacacaca cgtctaccca caggcacaca acagatggac
2700acgcgtacac agacatgcac acacccacag gcacaacacg tgcgcatgcc ggccggcccc
2760cgcccacatt ctcccagggc cctgccggat actctgtccc tgcagcagtt tgctccctgc
2820gctgtgctgg ccccggggct ttgggcccag gctctgcttg tccttctgtc tctgcttgga
2880ggtgctgcca tggcacccag cttgggctct gcctggggag cggaggcccc agggatagca
2940tgtgacccct gctgaggcca ggctcctgat gaaggcagca gatagccccc acacccaccg
3000gtgagcagaa ccagagcctg tgccatgtgc tgagagcagg cagtgactaa gcatatgggc
3060ccagagggca gagtggctgc cctgggcagc tgctcctctt agcgggaggc ctcaggagat
3120gagctagagc aagtctgccc ctgcaaatac cacctgctcc ccaacccaca gcagggagca
3180ggcgaggtca gacagcagca gcccgggaag gaccgagccc cagcagggaa ggcagggccc
3240gagtgaggtc tccacaccca acgcacagtg ctgtctctaa ctggggccac ctccgagtcc
3300ccgccacact cttggccctt tggagtcctg ggctccaggt gtctcccaag ggcccatctg
3360tgcaggggat gcaacccccc gaatgtcctc atcccactgt ggagctcagg tctctgtctg
3420ctccctgggt cctggcaggg taggacaagt ccgccaggat gtccccatgc agactctgct
3480ccaagaggga gctggagagt cagggccttg gtgagggagt caggatcggg ttccccccag
3540ctcagtcctc ccacctgcca gcccccacag cacagggcag ggccacaccc cctgcttccc
3600cctccaggag agtcaggaca tgctggccgc tgctccgctg gggccccgcc ctccagcccc
3660caccttggtc tgtgtgctgc atcccccacg ctctctctgc caccccagga ctctgaggaa
3720aagacctcag agtcccagcc ctgcccagtc tcggcctgtg cccccgctgc atcaggcttt
3780caggggccca gcccatgccc tgggcagtgc ccgagccccc ctgcacttgc tctccccacc
3840cctgggtgca gcacagccta ggggccaagg gtgggcctag aggatgggcc ccgggggggc
3900tttgctgggt gccaccccag cctgacccta ttcccccgtg ctgtgtctcc tgcagagggg
3960gaggtgagcg ccgacgagga gggctttgag aacctgtggg ccaccgcctc caccttcatc
4020gtcctcttcc tcctgagcct cttctacagt accaccgtca ccttgttcaa ggtagcacgg
4080ctgtggcaca gggaggaggg tgcagggcga gtgtggggcc cagggagcag cctgggctgg
4140acgtctagcc cggaggcccc cacaccaccc cactgggtca tctctgcccc ggctcccttc
4200ccgaccacgg ggaaagcatt tcacactgtc tctgttgcct gtaggtgaaa tgatcccaac
4260agaagaacat cggagaccag agagaggaac tcaaaggggc gctgcctccg ggtctggggt
4320cctggcctgc gtggcctgtt ggcacgtgtt tctcttcccc gcccggcctc cagttgtgtg
4380ctctcacaca ggcttctttc tcgaccggca ggggctggct ggcttgcagg ccacgaggtg
4440gggctctacc ccacactgct ttgctgtgta tacgcttgtt gcctgaaata aa
449234492DNAartificial sequencesyntheric polynucleotide (human Cepsilon
gene) 3tcagggagtg catccgcccc aacccttttc cccctcgtct cctgtgagaa ttccccgtcg
60gatacgagca gcgtggccgt tggctgcctc gcacaggact tccttcccga ctccatcact
120ttctcctgga aatacaagaa caactctgac atcagcagca cccggggctt cccatcagtc
180ctgagagggg gcaagtacgc agccacctca caggtgctgc tgccttccaa ggacgtcatg
240cagggcacag acgaacacgt ggtgtgcaaa gtccagcacc ccaacggcaa caaagaaaag
300aacgtgcctc ttccaggtga gggccgggcc cagccaccgg gacagagagg gagccgaagg
360ggggcgggag tggcgggcac cgggctgaca cgtgtccctc actgcagtga ttgctgagct
420gcctcccaaa gtgagcgtct tcgtcccacc ccgcgacggc ttcttcggca acccccgcaa
480gtccaagctc atctgccagg ccacgggttt cagtccccgg cagattcagg tgtcctggct
540gcgcgagggg aagcaggtgg ggtctggcgt caccacggac caggtgcagg ctgaggccaa
600agagtctggg cccacgacct acaaggtgac cagcacactg accatcaaag agagcgactg
660gctcagccag agcatgttca cctgccgcgt ggatcacagg ggcctgacct tccagcagaa
720tgcgtcctcc atgtgtgtcc ccggtgagtg acctgtcccc aggggcagca cccaccgaca
780cacaggggtc cactcgggtc tggcattcgc caccccggat gcagccatct actccctgag
840ccttggcttc ccagagcggc caagggcagg ggctcgggcg gcaggacccc tgggctcggc
900agaggcagtt gctactcttt gggtgggaac catgcctccg cccacatcca cacctgcccc
960acctctgact cccttctctt gactccagat caagacacag ccatccgggt cttcgccatc
1020cccccatcct ttgccagcat cttcctcacc aagtccacca agttgacctg cctggtcaca
1080gacctgacca cctatgacag cgtgaccatc tcctggaccc gccagaatgg cgaagctgtg
1140aaaacccaca ccaacatctc cgagagccac cccaatgcca ctttcagcgc cgtgggtgag
1200gccagcatct gcgaggatga ctggaattcc ggggagaggt tcacgtgcac cgtgacccac
1260acagacctgc cctcgccact gaagcagacc atctcccggc ccaagggtag gccccactct
1320tgcccctctt ctgcactcct gcaactcctt gcctctgggg gcatggtgga aagcacccct
1380cactcccccg ttgtctgggc aactggggaa aaggggactc aaccccagcc cacaggctgg
1440tccccccact gccccgccct caccaccatc tctgttcaca ggggtggccc tgcacaggcc
1500cgatgtctac ttgctgccac cagcccggga gcagctgaac ctgcgggagt cggccaccat
1560cacgtgcctg gtgacgggct tctctcccgc ggacgtcttc gtgcagtgga tgcagagggg
1620gcagcccttg tccccggaga agtatgtgac cagcgcccca atgcctgagc cccaggcccc
1680aggccggtac ttcgcccaca gcatcctgac cgtgtccgaa gaggaatgga acacggggga
1740gacctacacc tgcgtggtgg cccatgaggc cctgcccaac agggtcaccg agaggaccgt
1800ggacaagtcc accggtaaac ccaccctgta caacgtgtcc ctggtcatgt ccgacacagc
1860tggcacctgc tactgaccct gctggcctgc ccacaggctc ggggcggctg gccgctctgt
1920gtgtgcatgc aaactaaccg tgtcaacggg gtgagatgtt gcatcttata aaattagaaa
1980taaaaagatc cattcaaaag atactggtcc tgagtgcacg atgctctggc ctactggggc
2040ggcggctgtg ctgcacccac cctgcgcctc ccctgcagaa caccttcctc cacagccccc
2100acccctgcct cacccacctg cgtgcctcag tggcttctag aaacccctga attccctgca
2160gctgctcaca gcaggctgac ctcagacttg ccattcctcc tactgcttcc agaaagaaag
2220ctgaaagcaa ggccacacgt atacaggcag cacacaggca tgtgtggata cacatggaca
2280gacacggaca cacacaaaca catggacaca cagagacgtg ctaacccatg ggcacacaca
2340tacacagaca tggacccaca cacaaacata tgtggacaca catgtacaaa catgcacagg
2400cacacaaaga gaacactgac tacaggcaca cacacacacg ggcacacaca tggatatgtg
2460cacacatgga cacatacatg tgcaggacat gcacacacac agacacacta gcacagaggc
2520atacacacac agacacacac attcacaaac acacatgtgc atgcaaacac acacacatgt
2580acagacacga gtacatggac acatgcacac ccagagacac actgacacag acacacagga
2640gcatgtgata cactaacacg tggacacaca cgtctaccca caggcacaca acagatggac
2700acgcgtacac agacatgcac acacccacag gcacaacacg tgcgcatgcc ggccggcccc
2760cgcccacatt ctcccagggc cctgccggat actctgtccc tgcagcagtt tgctccctgc
2820gctgtgctgg ccccggggct ttgggcccag gctctgcttg tccttctgtc tctgcttgga
2880ggtgctgcca tggcacccag cttgggctct gcctggggag cggaggcccc agggatagca
2940tgtgacccct gctgaggcca ggctcctgat gaaggcagca gatagccccc acacccaccg
3000gtgagcagaa ccagagcctg tgccatgtgc tgagagcagg cagtgactaa gcatatgggc
3060ccagagggca gagtggctgc cctgggcagc tgctcctctt agcgggaggc ctcaggagat
3120gagctagagc aagtctgccc ctgcaaatac cacctgctcc ccaacccaca gcagggagca
3180ggcgaggtca gacagcagca gcccgggaag gaccgagccc cagcagggaa ggcagggccc
3240gagtgaggtc tccacaccca acgcacagtg ctgtctctaa ctggggccac ctccgagtcc
3300ccgccacact cttggccctt tggagtcctg ggctccaggt gtctcccaag ggcccatctg
3360tgcaggggat gcaacccccc gaatgtcctc atcccactgt ggagctcagg tctctgtctg
3420ctccctgggt cctggcaggg taggacaagt ccgccaggat gtccccatgc agactctgct
3480ccaagaggga gctggagagt cagggccttg gtgagggagt caggatcggg ttccccccag
3540ctcagtcctc ccacctgcca gcccccacag cacagggcag ggccacaccc cctgcttccc
3600cctccaggag agtcaggaca tgctggccgc tgctccgctg gggccccgcc ctccagcccc
3660caccttggtc tgtgtgctgc atcccccacg ctctctctgc caccccagga ctctgaggaa
3720aagacctcag agtcccagcc ctgcccagtc tcggcctgtg cccccgctgc atcaggcttt
3780caggggccca gcccatgccc tgggcagtgc ccgagccccc ctgcacttgc tctccccacc
3840cctgggtgca gcacagccta ggggccaagg gtgggcctag aggatgggcc ccgggggggc
3900tttgctgggt gccaccccag cctgacccta ttcccccgtg ctgtgtctcc tgcagagggg
3960gaggtgagcg ccgacgagga gggctttgag aacctgtggg ccaccgcctc caccttcatc
4020gtcctcttcc tcctgagcct cttctacagt accaccgtca ccttgttcaa ggtagcacgg
4080ctgtggcaca gggaggaggg tgcagggcga gtgtggggcc cagggagcag cctgggctgg
4140acgtctagcc cggaggcccc cacaccaccc cactgggtca tctctgcccc ggctcccttc
4200ccgaccacgg ggaaagcatt tcacactgtc tctgttgcct gtaggtgaaa tgatcccaac
4260agaagaacat cggagaccag agagaggaac tcaaaggggc gctgcctccg ggtctggggt
4320cctggcctgc gtggcctgtt ggcacgtgtt tctcttcccc gcccggcctc cagttgtgtg
4380ctctcacaca ggcttctttc tcgaccggca ggggctggct ggcttgcagg ccacgaggtg
4440gggctctacc ccacactgct ttgctgtgta tacgcttgtt gcctgaaata aa
44924894DNAartificial sequencesynthetic polynucleotide (neomycin
resistance gene) 4atgggatcgg ccattgaaca agatggattg cacgcaggtt
ctccggccgc ttgggtggag 60aggctattcg gctatgactg ggcacaacag acaatcggct
gctctgatgc cgccgtgttc 120cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
ccgacctgtc cggtgccctg 180aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
ccacgacggg cgttccttgc 240gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
ggctgctatt gggcgaagtg 300ccggggcagg atctcctgtc atctcacctt gctcctgccg
agaaagtatc catcatggct 360gatgcaatgc ggcggctgca tacgcttgat ccggctacct
gcccattcga ccaccaagcg 420aaacatcgca tcgagcgagc acgtactcgg atggaagccg
gtcttgtcga tcaggatgat 480ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
tcgccaggct caaggcgcgc 540atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
cctgcttgcc gaatatcatg 600gtggaaaatg gccgcttttc tggattcatc gactgtggcc
ggctgggtgt ggcggaccgc 660tatcaggaca tagcgttggc tacccgtgat attgctgaag
agcttggcgg cgaatgggct 720gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
cgcagcgcat cgccttctat 780cgccttcttg acgagttctt ctgaggggat cggcaataaa
aagacagaat aaaacgcacg 840ggtgttgggt cgtttgttcg gatcagcttc cgatcatatt
caataaccct taat 894534DNAartificial sequencesynthetic
oligonucleotide (loxP) 5ataacttcgt ataatgtatg ctatacgaag ttat
34615525DNAartificial sequencesynthetic
polynucleotide (transgenic DNA construct) 6gaagaggagt ttacgtccag
ccttcgaagg gtcctcaggg agtgcatccg ccccaaccct 60tttccccctc gtctcctgtg
agaattcccc gtcggatacg agcagcgtgg ccgttggctg 120cctcgcacag gacttccttc
ccgactccat cactttctcc tggaaataca agaacaactc 180tgacatcagc agcacccggg
gcttcccatc agtcctgaga gggggcaagt acgcagccac 240ctcacaggtg ctgctgcctt
ccaaggacgt catgcagggc acagacgaac acgtggtgtg 300caaagtccag caccccaacg
gcaacaaaga aaagaacgtg cctcttccag gtgagggccg 360ggcccagcca ccgggacaga
gagggagccg aaggggggcg ggagtggcgg gcaccgggct 420gacacgtgtc cctcactgca
gtgattgctg agctgcctcc caaagtgagc gtcttcgtcc 480caccccgcga cggcttcttc
ggcaaccccc gcaagtccaa gctcatctgc caggccacgg 540gtttcagtcc ccggcagatt
caggtgtcct ggctgcgcga ggggaagcag gtggggtctg 600gcgtcaccac ggaccaggtg
caggctgagg ccaaagagtc tgggcccacg acctacaagg 660tgaccagcac actgaccatc
aaagagagcg actggctcag ccagagcatg ttcacctgcc 720gcgtggatca caggggcctg
accttccagc agaatgcgtc ctccatgtgt gtccccggtg 780agtgacctgt ccccaggggc
agcacccacc gacacacagg ggtccactcg ggtctggcat 840tcgccacccc ggatgcagcc
atctactccc tgagccttgg cttcccagag cggccaaggg 900caggggctcg ggcggcagga
cccctgggct cggcagaggc agttgctact ctttgggtgg 960gaaccatgcc tccgcccaca
tccacacctg ccccacctct gactcccttc tcttgactcc 1020agatcaagac acagccatcc
gggtcttcgc catcccccca tcctttgcca gcatcttcct 1080caccaagtcc accaagttga
cctgcctggt cacagacctg accacctatg acagcgtgac 1140catctcctgg acccgccaga
atggcgaagc tgtgaaaacc cacaccaaca tctccgagag 1200ccaccccaat gccactttca
gcgccgtggg tgaggccagc atctgcgagg atgactggaa 1260ttccggggag aggttcacgt
gcaccgtgac ccacacagac ctgccctcgc cactgaagca 1320gaccatctcc cggcccaagg
gtaggcccca ctcttgcccc tcttctgcac tcctgcaact 1380ccttgcctct gggggcatgg
tggaaagcac ccctcactcc cccgttgtct gggcaactgg 1440ggaaaagggg actcaacccc
agcccacagg ctggtccccc cactgccccg ccctcaccac 1500catctctgtt cacaggggtg
gccctgcaca ggcccgatgt ctacttgctg ccaccagccc 1560gggagcagct gaacctgcgg
gagtcggcca ccatcacgtg cctggtgacg ggcttctctc 1620ccgcggacgt cttcgtgcag
tggatgcaga gggggcagcc cttgtccccg gagaagtatg 1680tgaccagcgc cccaatgcct
gagccccagg ccccaggccg gtacttcgcc cacagcatcc 1740tgaccgtgtc cgaagaggaa
tggaacacgg gggagaccta cacctgcgtg gtggcccatg 1800aggccctgcc caacagggtc
accgagagga ccgtggacaa gtccaccggt aaacccaccc 1860tgtacaacgt gtccctggtc
atgtccgaca cagctggcac ctgctactga ccctgctggc 1920ctgcccacag gctcggggcg
gctggccgct ctgtgtgtgc atgcaaacta accgtgtcaa 1980cggggtgaga tgttgcatct
tataaaatta gaaataaaaa gatccattca aaagatactg 2040gtcctgagtg cacgatgctc
tggcctactg gggcggcggc tgtgctgcac ccaccctgcg 2100cctcccctgc agaacacctt
cctccacagc ccccacccct gcctcaccca cctgcgtgcc 2160tcagtggctt ctagaaaccc
ctgaattccc tgcagctgct cacagcaggc tgacctcaga 2220cttgccattc ctcctactgc
ttccagaaag aaagctgaaa gcaaggccac acgtatacag 2280gcagcacaca ggcatgtgtg
gatacacatg gacagacacg gacacacaca aacacatgga 2340cacacagaga cgtgctaacc
catgggcaca cacatacaca gacatggacc cacacacaaa 2400catatgtgga cacacatgta
caaacatgca caggcacaca aagagaacac tgactacagg 2460cacacacaca cacgggcaca
cacatggata tgtgcacaca tggacacata catgtgcagg 2520acatgcacac acacagacac
actagcacag aggcatacac acacagacac acacattcac 2580aaacacacat gtgcatgcaa
acacacacac atgtacagac acgagtacat ggacacatgc 2640acacccagag acacactgac
acagacacac aggagcatgt gatacactaa cacgtggaca 2700cacacgtcta cccacaggca
cacaacagat ggacacgcgt acacagacat gcacacaccc 2760acaggcacaa cacgtgcgca
tgccggccgg cccccgccca cattctccca gggccctgcc 2820ggatactctg tccctgcagc
agtttgctcc ctgcgctgtg ctggccccgg ggctttgggc 2880ccaggctctg cttgtccttc
tgtctctgct tggaggtgct gccatggcac ccagcttggg 2940ctctgcctgg ggagcggagg
ccccagggat agcatgtgac ccctgctgag gccaggctcc 3000tgatgaaggc agcagatagc
ccccacaccc accggtgagc agaaccagag cctgtgccat 3060gtgctgagag caggcagtga
ctaagcatat gggcccagag ggcagagtgg ctgccctggg 3120cagctgctcc tcttagcggg
aggcctcagg agatgagcta gagcaagtct gcccctgcaa 3180ataccacctg ctccccaacc
cacagcaggg agcaggcgag gtcagacagc agcagcccgg 3240gaaggaccga gccccagcag
ggaaggcagg gcccgagtga ggtctccaca cccaacgcac 3300agtgctgtct ctaactgggg
ccacctccga gtccccgcca cactcttggc cctttggagt 3360cctgggctcc aggtgtctcc
caagggccca tctgtgcagg ggatgcaacc ccccgaatgt 3420cctcatccca ctgtggagct
caggtctctg tctgctccct gggtcctggc agggtaggac 3480aagtccgcca ggatgtcccc
atgcagactc tgctccaaga gggagctgga gagtcagggc 3540cttggtgagg gagtcaggat
cgggttcccc ccagctcagt cctcccacct gccagccccc 3600acagcacagg gcagggccac
accccctgct tccccctcca ggagagtcag gacatgctgg 3660ccgctgctcc gctggggccc
cgccctccag cccccacctt ggtctgtgtg ctgcatcccc 3720cacgctctct ctgccacccc
aggactctga ggaaaagacc tcagagtccc agccctgccc 3780agtctcggcc tgtgcccccg
ctgcatcagg ctttcagggg cccagcccat gccctgggca 3840gtgcccgagc ccccctgcac
ttgctctccc cacccctggg tgcagcacag cctaggggcc 3900aagggtgggc ctagaggatg
ggccccgggg gggctttgct gggtgccacc ccagcctgac 3960cctattcccc cgtgctgtgt
ctcctgcaga gggggaggtg agcgccgacg aggagggctt 4020tgagaacctg tgggccaccg
cctccacctt catcgtcctc ttcctcctga gcctcttcta 4080cagtaccacc gtcaccttgt
tcaaggtagc acggctgtgg cacagggagg agggtgcagg 4140gcgagtgtgg ggcccaggga
gcagcctggg ctggacgtct agcccggagg cccccacacc 4200accccactgg gtcatctctg
ccccggctcc cttcccgacc acggggaaag catttcacac 4260tgtctctgtt gcctgtaggt
gaaatgatcc caacagaaga acatcggaga ccagagagag 4320gaactcaaag gggcgctgcc
tccgggtctg gggtcctggc ctgcgtggcc tgttggcacg 4380tgtttctctt ccccgcccgg
cctccagttg tgtgctctca cacaggcttc tttctcgacc 4440ggcaggggct ggctggcttg
caggccacga ggtggggctc taccccacac tgctttgctg 4500tgtatacgct tgttgcctga
aataaatatg cacattttat ccatgaaact gctttctggt 4560gagggtttgt ttctttttca
aaactttcct gctacagggc attcaagcca tcgatgtcga 4620ggaattccga tcatattcaa
taacccttaa tataacttcg tataatgtat gctatacgaa 4680gttattaggt ctgaagagga
gtttacgtcc agccaagcta gcttggctgc aggtcgagca 4740gtgtggtttt caagaggaag
caaaaagcct ctccacccag gcctggaatg tttccaccca 4800atgtcgagca gtgtggtttt
gcaagaggaa gcaaaaagcc tctccaccca ggcctggaat 4860gtttccaccc aatgtcgagc
agtgtggttt tgcaagagga agcaaaaagc ctctccaccc 4920aggcctggaa tgtttccacc
caatgtcgag caaaccccgc ccagcgtctt gtcattggcg 4980aattcgaaca cgcagatgca
gtcggggcgg cgcggtcccc aggtccactt cgcatattaa 5040ggtgacgcgt gtggcctcga
acaccgagcg accctgcagc caatatggga tcggccattg 5100aacaagatgg attgcacgca
ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 5160actgggcaca acagacaatc
ggctgctctg atgccgccgt gttccggctg tcagcgcagg 5220ggcgcccggt tctttttgtc
aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 5280aggcagcgcg gctatcgtgg
ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 5340ttgtcactga agcgggaagg
gactggctgc tattgggcga agtgccgggg caggatctcc 5400tgtcatctca ccttgctcct
gccgagaaag tatccatcat ggctgatgca atgcggcggc 5460tgcatacgct tgatccggct
acctgcccat tcgaccacca agcgaaacat cgcatcgagc 5520gagcacgtac tcggatggaa
gccggtcttg tcgatcagga tgatctggac gaagagcatc 5580aggggctcgc gccagccgaa
ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 5640atctcgtcgt gacccatggc
gatgcctgct tgccgaatat catggtggaa aatggccgct 5700tttctggatt catcgactgt
ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 5760tggctacccg tgatattgct
gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 5820tttacggtat cgccgctccc
gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 5880tcttctgagg ggatcggcaa
taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg 5940ttcggatcag cttccgatca
tattcaataa cccttaatat aacttcgtat aatgtatgct 6000atacgaagtt attaggtctg
aagaggagtt tacgtccagc caagctaact tggcgccccc 6060actagggtcg aggagcttgg
taccgagctc ggatccacta gtaacggccg ccagtgtgct 6120ggaattcgcc cttgatgaat
gagcctggca tctggctccc tgccacgggg tccccagctc 6180ccccatccag gccccccagg
cctgatgggc gctggcctga ggctggcact gactaggttc 6240tgtcctcaca gcctccacac
agagcccatc cgtcttcccc ttgacccgct gctgcaaaaa 6300cattccctcc aatgccacct
ccgtgactct gggctgcctg gccacgggct acttcccgga 6360gccggtgatg gtgacctggg
acacaggctc cctcaacggg acaactatga ccttaccagc 6420caccaccctc acgctctctg
gtcactatgc caccatcagc ttgctgaccg tctcgggtgc 6480gtgggccaag cagatgttca
cctgccgtgt ggcacacact ccatcgtcca cagactgggt 6540cgacaacaaa accttcagcg
gtaagagagg gccaagctca gagaccacag ttcccaggag 6600tgccaggctg agggctggca
gagtgggcag gggttgaggg ggtgggtggg ctcaaacgtg 6660ggaacaccca gcatgcctgg
ggacccgggc caggacgtgg gggcaagagg agggcacaca 6720gagctcagag agcccaacaa
ccctcatgac caccagctct cccccagtct gctccaggga 6780cttcaccccg cccaccgtga
agatcttaca gtcgtcctgc gacggcggcg ggcacttccc 6840cccgaccatc cagctcctgt
gcctcgtctc tgggtacacc ccagggacta tcaacatcac 6900ctggctggag gacgggcagg
tcatggacgt ggacttgtcc accgcctcta ccacgcagga 6960gggtgagctg gcctccacac
aaagcgagct caccctcagc cagaagcact ggctgtcaga 7020ccgcacctac acctgccagg
tcacctatca aggtcacacc tttgaggaca gcaccaagaa 7080gtgtgcaggt acgttcccac
ctgccctggt ggccgccacg gaggccagag aagaggggcg 7140ggtgggcctc acacagccct
ccggtgtacc acagattcca acccgagagg ggtgagcgcc 7200tacctaagcc ggcccagccc
gttcgacctg ttcatccgca agtcgcccac gatcacctgt 7260ctggtggtgg acctggcacc
cagcaagggg accgtgaacc tgacctggtc ccgggccagt 7320gggaagcctg tgaaccactc
caccagaaag gaggagaagc agcgcaatgg cacgttaacc 7380gtcacgtcca ccctgccggt
gggcacccga gactggatcg agggggagac ctaccaatgc 7440agggtgaccc acccccacct
gcccagggcc ctcatgcggt ccacgaccaa gaccagcggt 7500gagccatggg caggccgggg
tcgtggggga agggagggag cgagtgagcg gggcccgggc 7560tgaccccacg tctggccaca
ggcccgcgtg ctgccccgga agtctatgcg tttgcgacgc 7620cggagtggcc ggggagccgg
gacaagcgca ccctcgcctg cctgatccag aacttcatgc 7680ctgaggacat ctcggtgcag
tggctgcaca acgaggtgca gctcccggac gcccggcaca 7740gcacgacgca gccccgcaag
accaagggct ccggcttctt cgtcttcagc cgcctggagg 7800tgaccagggc cgaatgggag
cagaaagatg agttcatctg ccgtgcagtc catgaggcag 7860cgagcccctc acagaccgtc
cagcgagcgg tgtctgtaaa tcccggtaaa tgacgtactc 7920ctgcctccct ccctcccagg
gctccatcca gctgtgcagt ggggaggact ggccagacct 7980tctgtccact gttgcaatga
ccccaggaag ctacccccaa taaactgtgc ctgctcagag 8040ccccaggtac acccattctt
gggagcgggc agggctgtgg gcaggtgcat cttggcacag 8100aggaatgggc cccccaggag
gggcagtggg aggaggtggg cagggctgag tccccccagg 8160agaggtggtg ggaggaggtg
ggcaggggtg aggtgccact catccatctg ccttcgtgtc 8220agggttattt gtcaaacagc
atatctgcag ggactcatca cagctacccc gggcctctct 8280gcccccactc tgggtctacc
ccctccaagg agtccaaaga cccaggggag gtcctcaggg 8340aaggggcaag ggagccccca
cagccctccc tcttgggggc ttggcttcta cccccctgga 8400caggagcccc tgcaccccca
ggtatagatg ggcacacagg cccctccagg tggaaaaaca 8460gccctaagtg aaacccccac
acagacacac acaacccgac agccctcgcc caagtctgtg 8520ccactggcgt tcgcctctct
gccctgtccc gccttgccga gtcctggccc cagcaccggg 8580gccggtggag ccgagcccac
tcacaccccg cagcctccgc caccctgccc tgtgggcaca 8640ccaggcccag gtcagagcca
ggccccctct cctactgccc cccaccgccc cttggtccat 8700cctgaatcgg cctccagggg
atcgccagcc tcacacaccc ggtctcgccc actcacgcct 8760cactcaaggc acagctgtgc
acacactagg ccccatagca actccacagc accctgtacc 8820accaccaggg cgccatagac
accccacacg tggtcacacg tggcccacac tccgcctctc 8880acgctgcctc cagccaggct
actgccaagc ccttcctctg agccatacct gggccgctgg 8940atcccagaga gaaatggaga
ggccctcacg tggtgtcctc cagtccaacc ctccctgtca 9000ccctgtcagc agcaccccac
agccaaacac aggatggatg cgtgggctcc atcccccact 9060cacccacacc tgaaccccag
agcaggctac gtgcccctca cagacctcaa acccacatgt 9120gcatctgaca ccccagatcc
aaacgctccc cccggtcatg cacaccaagg gcacagcacc 9180caccaaatcc acacggaaac
acgggcaccg ggcaccccat gagcacaaag cccctccatg 9240tctgaagaca gtccctgcac
accgtcacag ccatacattc agcttcactc tcacgtccca 9300gcccacctgc acccagctct
gggcctggag cagcagaaag aggtgtgagg gcccgaggcc 9360ggacctgcac ctgctgatga
cccgggacca gcaggcagct cacggtgttg gggaagggag 9420tggagggcac ccagggcagg
agccagaggg accaggctgg tgggcggggc cgggccgggg 9480tagggccagg aggcagctct
ggacacccac aggcctgggc tcatagtcca caccaggaca 9540gcccctcaga gcacccatgc
agtgagtccc aggtcttggg agccaggccg cagagctcac 9600gcatccttcc gagggccctg
agtgaggcgg ccactgctgc gccgaggggt tgggtccttc 9660tctggggagg gcgtggggtc
tagagaggcg gagtggaggt aaccagaggt caggagagaa 9720gccgtaagga acagagggaa
aatggggcca gagtcggggc gcagggacga gaggtcagga 9780gtggtcggcc tggccctggg
ccgttgactg actcgggacc tgggtgccca ccctcagggc 9840tggctggcgg ctccgcgcag
tcccagaggg ccccggatag ggtgctctgc cactccggac 9900agcagcaggg actgccgaga
gcggcaggag gctctgtccc ccacccccgc tgccactgtg 9960gagccgggag ggctgactgg
ccaggtcccc cagagctgga cgtgtgcgtg gaggaggccg 10020agggcgaggc gccgtggacg
tggaccggcc tctgcatctt cgccgcactc ttcctgctca 10080gcgtgagcta cagcgccgcc
ctcacgctcc tcatggtggg cacccacctc caggggccca 10140gccagggcag ggggttgggc
agagccagca gagcgccctg acccacgccc tcccctcagg 10200tgcagcggtt cctctcagcc
acgcggcagg ggaggcccca gacctccctc gactacacca 10260acgtcctcca gccccacgcc
taggccgcgg gccactcacg ctccaccagg cccagctttt 10320tctctgccag cgcctgagcc
tccctcgggc tgcaccctgc cctgggtggg aaaagggaag 10380cagacaagaa aagggggcac
aaggtcacta ctgtgggctg atggccagtg aacctgagcc 10440cagaggggcc ggctcagccg
caaggttaca ggcgccgaga gaaccaccag tcgcagcccc 10500cacccgaaaa ccgtgtctgt
cccttcaaca gagtcatcga ggaggggtgg ctgctagccg 10560ttctgagctc atcgaagggc
gaattctgca gatatccatc acactggcgg ccgctcgacc 10620taggatctcc tgtctgacag
gaggcaagaa gacagattct tacccctcca tttctctttt 10680atccctctct ggtcctcaga
gagtcagtcc ttcccaaatg tcttccccct cgtctcctgc 10740gagagccccc tgtctgataa
gaatctggtg gccatgggct gcctggcccg ggacttcctg 10800cccagcacca tttccttcac
ctggaactac cagaacaaca ctgaagtcat ccagggtatc 10860agaaccttcc caacactgag
gacagggggc aagtacctag ccacctcgca ggtgttgctg 10920tctcccaaga gcatccttga
aggttcagat gaatacctgg tatgcaaaat ccactacgga 10980ggcaaaaaca gagatctgca
tgtgcccatt ccaggtaaga accaaaccct cccagcaggg 11040gtgcccaggc ccaggcatgg
cccagaggga gcagcgggtg gggcttaggc caagctgagc 11100tcacaccttg acctttcatt
ccagctgtcg cagagatgaa ccccaatgtg ttcgtcccac 11160cacgggatgg cttctctggc
cctgcaccac gcaagtctaa actcatctgc gaggccacga 11220acttcactcc aaaaccgatc
acagtatcct ggctaaagga tgggaagctc gtggaatctg 11280gcttcaccac agatccggtg
accatcgaga acaaaggatc cacaccccaa acctacaagg 11340tcataagcac acttaccatc
tctgaaatcg actggctgaa cctgaatgtg tacacctgcc 11400gtgtggatca caggggtctc
accttcttga agaacgtgtc ctccacatgt gctgccagtg 11460agtggcctgg gataagccca
atgcctagcc ctcccagatt agggaagtcc tcctacaatt 11520atggccaatg ccacccagac
atggtcattt gctccttgaa ctttggctcc ccagagtggc 11580caaggacaag aatgagcaat
aggcagtaga ggggtgagaa tcagctggaa ggaccagcat 11640cttcccttaa gtaggtttgg
gggatggaga ctaagctttt ttccaacttc acaactagat 11700atgtcataac ctgacacagt
gttctcttga ctgcaggtcc ctccacagac atcctaacct 11760tcaccatccc cccctccttt
gccgacatct tcctcagcaa gtccgctaac ctgacctgtc 11820tggtctcaaa cctggcaacc
tatgaaaccc tgaatatctc ctgggcttct caaagtggtg 11880aaccactgga aaccaaaatt
aaaatcatgg aaagccatcc caatggcacc ttcagtgcta 11940agggtgtggc tagtgtttgt
gtggaagact ggaataacag gaaggaattt gtgtgtactg 12000tgactcacag ggatctgcct
tcaccacaga agaaattcat ctcaaaaccc aatggtaggt 12060atcccccctt cccttcccct
ccaattgcag gacccttcct gtacctcata gggagggcag 12120gtcctcttcc accctatcct
cactactgtc ttcatttaca gaggtgcaca aacatccacc 12180tgctgtgtac ctgctgccac
cagctcgtga gcaactgaac ctgagggagt cagccacagt 12240cacctgcctg gtgaagggct
tctctcctgc agacatcagt gtgcagtggc ttcagagagg 12300gcaactcttg ccccaagaga
agtatgtgac cagtgccccg atgccagagc ctggggcccc 12360aggcttctac tttacccaca
gcatcctgac tgtgacagag gaggaatgga actccggaga 12420gacctatacc tgtgttgtag
gccacgaggc cctgccacac ctggtgaccg agaggaccgt 12480ggacaagtcc actggtaaac
ccacactgta caatgtctcc ctgatcatgt ctgacacagg 12540cggcacctgc tattgaccat
gctagcgctc aaccaggcag gccctgggtg tccagttgct 12600ctgtgtatgc aaactaacca
tgtcagagtg agatgttgca ttttataaaa attagaaata 12660aaaaaaatcc attcaaacgt
cactggtttt gattatacaa tgctcatgcc tgctgagaca 12720gttgtgtttt gcttgctctg
cacacaccct gcatacttgc ctccaccctg gcccttcctc 12780taccttgcca gtttcctcct
tgtgtgtgaa ctcagtcagg cttacaacag acagagtatg 12840aacatgcgat tcctccagct
acttcttgat atatggctga aagcttgcct aacctggtgc 12900aggcagcatt caggcacata
tatagacaca catgcattta tacatagata tataggtaca 12960catgtgtaga cacatacatg
aatgtgtatt catggacaca cagacaaagg tacacatata 13020tacacatgag ttcatgcgca
cacacatgca tggacactta caaacgcctt cagagacaaa 13080taggcataga cacacaacca
ctcacagaaa cagataccaa tatgcatggt cctgtgtaca 13140cagaaacaga ctataggcaa
atatacacaa ataaactata tagatacaaa gatatgcata 13200tacacacatg tacagaaaca
tcttcacatg tgtacactaa catgtggaca ggtatagcac 13260acagatacac ctggactctg
accagggctg taatctccaa ggctcacggc tcagagagcc 13320tacactaggc tgggtcactg
atactcctca ggagcccact ctatgattgg gagagataac 13380cccaggtaca aagtatgcct
atctgtctca acaccatggg gcagaagata ctccactaac 13440cacccatgac agaaagttag
ccttggctgt gtctccatta atagaacacc tcagaagacc 13500aatgtgaaat tgcctaaccc
actcacaccc accctgatct ccagttcaaa atgcagaaaa 13560cataatgcag ttgtccaaaa
gatgccccaa ccacacacac acacacacac acacacacac 13620acacacacac acacacacac
acacacacac accatcaagg agcctctgta aggagtcacc 13680acccaataac actgcctctt
tgggctcata tcctggacat tcttcatatt catatccatt 13740tggggcctag gctttagata
tccccaaggg ctcatcttta cagggatcag agatcccaat 13800aaatgccctg gtcccacagc
ctccctcagg tatctgtctg tttatctctt ggtaccaaga 13860cccaacattg ctggcagggg
taggacaagc aacgcacggg aactctgatc aaagaaagtc 13920atgagatgcc tgagtccttc
aggaagtaag gagggacaac ctctggtatc cctgttctta 13980ttgctaaagc ccaagagaca
gggagacctg ctctaaattc tcagtctaaa cagcaccgat 14040ggcaccacct gctcagggaa
agtccagagc acaccaatat cattttgcca cagttcctga 14100gtctgccttt acccaggtcc
atacattgca tctgtcttgc ttgctctgct gccccagggc 14160tcctggaaca aaggctccaa
attagtgtgt cctacagctt ggcctgttct gtgcctccgt 14220ctagcttgag ctattagggg
accagtcaat actcgctaag attctccaga accatcaggg 14280caccccaacc cttatgcaaa
tgctcagtca ccccaagact tggcttgacc ctccctctct 14340gtgtcccttc atagaggggg
aggtgaatgc tgaggaggaa ggctttgaga acctgtggac 14400cactgcctcc accttcatcg
tcctcttcct cctgagcctc ttctacagca ccaccgtcac 14460cctgttcaag gtagtatggt
tgtggggctg aggacacagg gctgggacag ggagtcacca 14520gtcctcactg cctctacctc
tactccctac aagtggacag caattcacac tgtctctgtc 14580acctgcaggt gaaatgactc
tcagcatgga aggacagcag agaccaagag atcctcccac 14640agggacacta cctctgggcc
tgggatacct gactgtatga ctagtaaact tattcttacg 14700tctttcctgt gttgccctcc
agcttttatc tctgagatgg tcttctttct agactgacca 14760aagacttttt gtcaacttgt
acaatctgaa gcaatgtctg gcccacagac agctgagctg 14820taaacaaatg tcacatggaa
ataaatactt tatcttgtga actcacttta ttgtgaagga 14880atttgttttg tttttcaaac
ctttcctgcg gtgttgacag cccaaggatt atctgaatag 14940agcttaggaa ctggaaatgg
aacagtgcag tctgatggta cttaagggag aaagagggaa 15000aggaggtgtg gaagaagaaa
aaagagaagc agagggggag gggagaaggg agagggagag 15060ggagagggag agggagaggg
agagggagag ggagagagag agagagagag agagagagag 15120agagagcatg cactctaaca
gcaaagtaca acacaggcag ccaatggata acactctggt 15180tatctaccct gatggaagaa
gggaagtagg gcagagaaaa ttccaggcct aatctcccaa 15240aagcaacaga acctggaaac
tagcctctag ccttaggtct ctgctctgtc cccagcccac 15300catcttgggc tggtgttgct
tcaagctagt aatttaggtc ttatcccaaa gctttgtggt 15360atgtgggtgt gcctttgggg
agttggctga gattttgaag atgtttgtac ctctcccaca 15420acatgacaag ccctaggggt
tagtcaataa ctcaaattct ctgtctatga caactgctgt 15480atgactatat gaagaaatgg
gataaagatg ctatagtcac tcgag 1552575270DNAartificial
sequencesynthetic polynucleotide (5' arm) 7agcgcgcgta atacgactca
ctatagggcg aattggagct ccaccgcggt ggcggccgct 60cctgagagaa cagactctgg
aaatagatgg gacttaagga gctaagatct agagctcatc 120tacagagcag aatcccagcc
aagagaacaa agaatactgg ctctctctcc tgttccctac 180tcctagagtt ctaaaacaca
ctatagggaa gggagcctct agacctccgt ccattcccca 240tcttgctcat tccatcttcc
catgtcccca ggtctccaag ccacagacac tacctttcct 300attcacccac ctttctgtgt
ccctaggtcc ccaggccata gtcacctccc cccacacaca 360ccccactcac cctgccccat
ctatgcccct agatgcttac ttaccagagt cttttgtctg 420acgtggggct acaagcatct
atgctcccta agcacctact gctgacctgt aggacccagc 480tctgaaccaa ctcatataag
taaatacaga ctctcccctg tcttaggatg gcctcctgga 540tcaggaggag accactgcca
aagaaccttc tctcagagca ctgaactcct cccctgtacc 600acttaggaca gacctgagac
ctattattac tgattaccag agctctggca gtgaccacgg 660aggagatagg tccaccctgg
acacaggaaa cacagcagca gagatactgc tccatcacaa 720cagtagagtg acactttaga
ctttaatttg ggtcactttc ctgctgcaga ggtgggatca 780gaaagcaaag agcagtatga
gtgcctgata ggcacccaag tacactatag agtactcatg 840gtgaataagg tacctccatg
gcttcccagg gaggggcagc cccaccccca ccatcacaga 900cctttctcca tagttgataa
ctcagacaca agtgaatgac agatggacct ccatctactc 960ttattttaaa aagaagacaa
accccacagg ctcgagaact ttagcgactg ttttgagaga 1020aatcattggt ccctgactca
agagatgact ggcagattgg ggatcagaat acccatactc 1080tgtggctagt gtgaggttta
agcctcagag tccctgtggt ctctgactgg tgcaaggttt 1140tgactaagcg gagcaccaca
gtgctaactg ggaccacggt gacacgtggc tcaacaaaaa 1200ccttctgttt ggagctctcc
aggggcagcc tgagctatga ggaagtagag aggcttgaga 1260aatctgagga agaaaagagt
agatctgaga ggaaaggtag ctttctggag gtcaggagac 1320agtgcagaga agaacgagtt
actgtggaca ggtcttagat ggggaaagaa tgagcaaatg 1380caagcatcag aagggtggat
gcaatgtcct gccaaggact taccaagagg atccccggac 1440agagcaggca ggtggagttg
actgagagga cagggtaggt gcaggtccct ctctcgtttc 1500ctttctcctt ctcctgtttc
cttcctctct tgtcacaggt ctcactatgc tagccaaggc 1560tagcctgaaa gattaccatc
ctacagatgg gcccatccag ttgagttaag gtggagatct 1620ctccaaacat ctgagtttct
gaggcttgga tgccactggg gacgccaagg gactttgggc 1680tgggtttggt tggccccaga
tgaagggcta cttcactggg tctataatta ctctgatgtc 1740taggaccagg gggctcaggt
cactcaggtc aggtgagtcc tgcatctggg gactgtgggg 1800ttcaggtgtc ctaaggcagg
atgtggagag agttttagta taggaacaga ggcagaacag 1860agactgtgct actggtactt
cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 1920ggtaagctgg cttttttctt
tctgcacatt ccattctgaa atgggaaaag atattctcag 1980atctccccat gtcaggccat
ctgccacact ctgcatgctg cagaagcttt tctgtaagga 2040tagggtcttc actcccagga
aaagaggcag tcagaggcta gctgcctgtg gaacagtgac 2100aatcatggaa aataggcatt
tacattgtta ggctacatgg gtagatgggt ttttgtacac 2160ccactaaagg ggtctatgat
agtgtgacta ctttgactac tggggccaag gcaccactct 2220cacagtctcc tcaggtgagt
ccttacaacc tctctcttct attcagctta aatagatttt 2280actgcatttg ttggggggga
aatgtgtgta tctgaatttc aggtcatgaa ggactaggga 2340caccttggga gtcagaaagg
gtcattggga gccctggctg acgcagacag acatcctcag 2400ctcccatact tcatggccag
agatttatag ggatcctggc cagcattgcc gctaggtccc 2460tctcttctat gctttctttg
tccctcactg gcctccatct gagataatcc tggagcccta 2520gccaaggatc atttattgtc
aggggtctaa tcattgttgt cacaatgtgc ctggtttgct 2580tactggggcc aagggactct
ggtcactgtc tctgcaggtg agtcctaact tctcccattc 2640taaatgcatg ttggggggat
tctgagcctt caggaccaag attctctgca aacgggaatc 2700aagattcaac ccctttgtcc
caaagttgag acatgggtct gggtcaggga ctctctgcct 2760gctggtctgt ggtgacatta
gaactgaagt atgatgaagg atctgccaga actgaagctt 2820gaagtctgag gcagaatctt
gtccagggtc tatcggactc ttgtgagaat taggggctga 2880cagttgatgg tgacaatttc
agggtcagtg actgtctggt ttctctgagg tgaggctgga 2940atataggtca ccttgaagac
ttaagagggg tccagggggc ttctgcacag gcagggaaca 3000gaatgtggaa caatgacttg
aatggttgat tcttgtgtga caccaggaat tggcataatg 3060tctgagttgc ccaggggtga
ttctagtcag actctggggt ttttgtcggg tatagaggaa 3120aaatccacta ttgtgattac
tatgctatgg actactgggg tcaaggaacc tcagtcaccg 3180tctcctcagg taagaatggc
ctctccaggt ctttattttt aacctttgtt atggagtttt 3240ctgagcattg cagactaatc
ttggatattt gtccctgagg gagccggctg agagaagttg 3300ggaaataaac tgtctaggga
tctcagagcc tttaggacag attatctcca catctttgaa 3360aaactaagaa tctgtgtgat
ggtgttggtg gagtccctgg atgatgggat agggactttg 3420gaggctcatt tgagggagat
gctaaaacaa tcctatggct ggagggatag ttggggctgt 3480agttggagat tttcagtttt
tagaataaaa gtattagctg cggaatatac ttcaggacca 3540cctctgtgac agcatttata
cagtatccga tgcataggga caaagagtgg agtggggcac 3600tttctttaga tttgtgagga
atgttccaca ctagattgtt taaaacttca tttgttggaa 3660ggagagctgt cttagtgatt
gagtcaaggg agaaaggcat ctagcctcgg tctcaaaagg 3720gtagttgctg tctagagagg
tctggtggag cctgcaaaag tccagctttc aaaggaacac 3780agaagtatgt gtatggaata
ttagaagatg ttgcttttac tcttaagttg gttcctagga 3840aaaatagtta aatactgtga
ctttaaaatg tgagagggtt ttcaagtact cattttttta 3900aatgtccaaa atttttgtca
atcaatttga ggtcttgttt gtgtagaact gacattactt 3960aaagtttaac cgaggaatgg
gagtgaggct ctctcataac ctattcagaa ctgactttta 4020acaataataa attaagtttc
aaatattttt aaatgaattg agcaatgttg agttggagtc 4080aagatggccg atcagaacca
gaacacctgc agcagctggc aggaagcagg tcatgtggca 4140aggctatttg gggaagggaa
aataaaacca ctaggtaaac ttgtagctgt ggtttgaaga 4200agtggttttg aaacactctg
tccagcccca ccaaaccgaa agtccaggct gagcaaaaca 4260ccacctgggt aatttgcatt
tctaaaataa gttgaggatt cagccgaaac tggagaggtc 4320ctcttttaac ttattgagtt
caacctttta attttagctt gagtagttct agtttcccca 4380aacttaagtt tatcgacttc
taaaatgtat ttagaattca ttttcaaaat taggttatgt 4440aagaaattga aggactttag
tgtctttaat ttctaatata tttagaaaac ttcttaaaat 4500tactctatta ttcttccctc
tgattattgg tctccattca attcttttcc aatacccgaa 4560gcatttacag tgactttgtt
catgatcttt tttagttgtt tgttttgcct tactattaag 4620actttgacat tctggtcaaa
acggcttcac aaatcttttt caagaccact ttctgagtat 4680tcattttagg agaaagactt
tttttttaaa tgaatgcaat tatctagact tatttcagtt 4740gaacatgctg gttggtggtt
gagaggacac tcagtcagtc agtggcgtga agggcttcta 4800agccagtcca catgctctgt
gtgaactccc tctggccctg cttattgttg aatgggccaa 4860aggtctgaga ccaggctgct
gctgggtagg cctggacttt gggtctccca cccagacctg 4920ggaatgtatg gttgtggctt
ctgccaccca tccacctggc tgctcatgga ccagccagcc 4980tcggtggctt tgaaggaaca
attccacaca aagactctgg acctctccga aaccaggcac 5040cgcaaatggt aagccagagg
cagccacagc tgtggctgct gctcttaaag cttgtaaact 5100gtttctgctt aagagggact
gagtcttcag tcattgcttt agggggagaa agagacattt 5160gtgtgtcttt tgagtaccgt
tgtctgggtc actcacattt aactttcctt gaaaaactag 5220actcgacatc gattgtcgag
gaattccgat catattcaat aacccttaat 527084902DNAartificial
sequencesynthetic polynucleotide (3'arm) 8gatctcctgt ctgacaggag
gcaagaagac agattcttac ccctccattt ctcttttatc 60cctctctggt cctcagagag
tcagtccttc ccaaatgtct tccccctcgt ctcctgcgag 120agccccctgt ctgataagaa
tctggtggcc atgggctgcc tggcccggga cttcctgccc 180agcaccattt ccttcacctg
gaactaccag aacaacactg aagtcatcca gggtatcaga 240accttcccaa cactgaggac
agggggcaag tacctagcca cctcgcaggt gttgctgtct 300cccaagagca tccttgaagg
ttcagatgaa tacctggtat gcaaaatcca ctacggaggc 360aaaaacagag atctgcatgt
gcccattcca ggtaagaacc aaaccctccc agcaggggtg 420cccaggccca ggcatggccc
agagggagca gcgggtgggg cttaggccaa gctgagctca 480caccttgacc tttcattcca
gctgtcgcag agatgaaccc caatgtgttc gtcccaccac 540gggatggctt ctctggccct
gcaccacgca agtctaaact catctgcgag gccacgaact 600tcactccaaa accgatcaca
gtatcctggc taaaggatgg gaagctcgtg gaatctggct 660tcaccacaga tccggtgacc
atcgagaaca aaggatccac accccaaacc tacaaggtca 720taagcacact taccatctct
gaaatcgact ggctgaacct gaatgtgtac acctgccgtg 780tggatcacag gggtctcacc
ttcttgaaga acgtgtcctc cacatgtgct gccagtgagt 840ggcctgggat aagcccaatg
cctagccctc ccagattagg gaagtcctcc tacaattatg 900gccaatgcca cccagacatg
gtcatttgct ccttgaactt tggctcccca gagtggccaa 960ggacaagaat gagcaatagg
cagtagaggg gtgagaatca gctggaagga ccagcatctt 1020cccttaagta ggtttggggg
atggagacta agcttttttc caacttcaca actagatatg 1080tcataacctg acacagtgtt
ctcttgactg caggtccctc cacagacatc ctaaccttca 1140ccatcccccc ctcctttgcc
gacatcttcc tcagcaagtc cgctaacctg acctgtctgg 1200tctcaaacct ggcaacctat
gaaaccctga atatctcctg ggcttctcaa agtggtgaac 1260cactggaaac caaaattaaa
atcatggaaa gccatcccaa tggcaccttc agtgctaagg 1320gtgtggctag tgtttgtgtg
gaagactgga ataacaggaa ggaatttgtg tgtactgtga 1380ctcacaggga tctgccttca
ccacagaaga aattcatctc aaaacccaat ggtaggtatc 1440cccccttccc ttcccctcca
attgcaggac ccttcctgta cctcataggg agggcaggtc 1500ctcttccacc ctatcctcac
tactgtcttc atttacagag gtgcacaaac atccacctgc 1560tgtgtacctg ctgccaccag
ctcgtgagca actgaacctg agggagtcag ccacagtcac 1620ctgcctggtg aagggcttct
ctcctgcaga catcagtgtg cagtggcttc agagagggca 1680actcttgccc caagagaagt
atgtgaccag tgccccgatg ccagagcctg gggccccagg 1740cttctacttt acccacagca
tcctgactgt gacagaggag gaatggaact ccggagagac 1800ctatacctgt gttgtaggcc
acgaggccct gccacacctg gtgaccgaga ggaccgtgga 1860caagtccact ggtaaaccca
cactgtacaa tgtctccctg atcatgtctg acacaggcgg 1920cacctgctat tgaccatgct
agcgctcaac caggcaggcc ctgggtgtcc agttgctctg 1980tgtatgcaaa ctaaccatgt
cagagtgaga tgttgcattt tataaaaatt agaaataaaa 2040aaaatccatt caaacgtcac
tggttttgat tatacaatgc tcatgcctgc tgagacagtt 2100gtgttttgct tgctctgcac
acaccctgca tacttgcctc caccctggcc cttcctctac 2160cttgccagtt tcctccttgt
gtgtgaactc agtcaggctt acaacagaca gagtatgaac 2220atgcgattcc tccagctact
tcttgatata tggctgaaag cttgcctaac ctggtgcagg 2280cagcattcag gcacatatat
agacacacat gcatttatac atagatatat aggtacacat 2340gtgtagacac atacatgaat
gtgtattcat ggacacacag acaaaggtac acatatatac 2400acatgagttc atgcgcacac
acatgcatgg acacttacaa acgccttcag agacaaatag 2460gcatagacac acaaccactc
acagaaacag ataccaatat gcatggtcct gtgtacacag 2520aaacagacta taggcaaata
tacacaaata aactatatag atacaaagat atgcatatac 2580acacatgtac agaaacatct
tcacatgtgt acactaacat gtggacaggt atagcacaca 2640gatacacctg gactctgacc
agggctgtaa tctccaaggc tcacggctca gagagcctac 2700actaggctgg gtcactgata
ctcctcagga gcccactcta tgattgggag agataacccc 2760aggtacaaag tatgcctatc
tgtctcaaca ccatggggca gaagatactc cactaaccac 2820ccatgacaga aagttagcct
tggctgtgtc tccattaata gaacacctca gaagaccaat 2880gtgaaattgc ctaacccact
cacacccacc ctgatctcca gttcaaaatg cagaaaacat 2940aatgcagttg tccaaaagat
gccccaacca cacacacaca cacacacaca cacacacaca 3000cacacacaca cacacacaca
cacacacacc atcaaggagc ctctgtaagg agtcaccacc 3060caataacact gcctctttgg
gctcatatcc tggacattct tcatattcat atccatttgg 3120ggcctaggct ttagatatcc
ccaagggctc atctttacag ggatcagaga tcccaataaa 3180tgccctggtc ccacagcctc
cctcaggtat ctgtctgttt atctcttggt accaagaccc 3240aacattgctg gcaggggtag
gacaagcaac gcacgggaac tctgatcaaa gaaagtcatg 3300agatgcctga gtccttcagg
aagtaaggag ggacaacctc tggtatccct gttcttattg 3360ctaaagccca agagacaggg
agacctgctc taaattctca gtctaaacag caccgatggc 3420accacctgct cagggaaagt
ccagagcaca ccaatatcat tttgccacag ttcctgagtc 3480tgcctttacc caggtccata
cattgcatct gtcttgcttg ctctgctgcc ccagggctcc 3540tggaacaaag gctccaaatt
agtgtgtcct acagcttggc ctgttctgtg cctccgtcta 3600gcttgagcta ttaggggacc
agtcaatact cgctaagatt ctccagaacc atcagggcac 3660cccaaccctt atgcaaatgc
tcagtcaccc caagacttgg cttgaccctc cctctctgtg 3720tcccttcata gagggggagg
tgaatgctga ggaggaaggc tttgagaacc tgtggaccac 3780tgcctccacc ttcatcgtcc
tcttcctcct gagcctcttc tacagcacca ccgtcaccct 3840gttcaaggta gtatggttgt
ggggctgagg acacagggct gggacaggga gtcaccagtc 3900ctcactgcct ctacctctac
tccctacaag tggacagcaa ttcacactgt ctctgtcacc 3960tgcaggtgaa atgactctca
gcatggaagg acagcagaga ccaagagatc ctcccacagg 4020gacactacct ctgggcctgg
gatacctgac tgtatgacta gtaaacttat tcttacgtct 4080ttcctgtgtt gccctccagc
ttttatctct gagatggtct tctttctaga ctgaccaaag 4140actttttgtc aacttgtaca
atctgaagca atgtctggcc cacagacagc tgagctgtaa 4200acaaatgtca catggaaata
aatactttat cttgtgaact cactttattg tgaaggaatt 4260tgttttgttt ttcaaacctt
tcctgcggtg ttgacagccc aaggattatc tgaatagagc 4320ttaggaactg gaaatggaac
agtgcagtct gatggtactt aagggagaaa gagggaaagg 4380aggtgtggaa gaagaaaaaa
gagaagcaga gggggagggg agaagggaga gggagaggga 4440gagggagagg gagagggaga
gggagaggga gagagagaga gagagagaga gagagagaga 4500gagcatgcac tctaacagca
aagtacaaca caggcagcca atggataaca ctctggttat 4560ctaccctgat ggaagaaggg
aagtagggca gagaaaattc caggcctaat ctcccaaaag 4620caacagaacc tggaaactag
cctctagcct taggtctctg ctctgtcccc agcccaccat 4680cttgggctgg tgttgcttca
agctagtaat ttaggtctta tcccaaagct ttgtggtatg 4740tgggtgtgcc tttggggagt
tggctgagat tttgaagatg tttgtacctc tcccacaaca 4800tgacaagccc taggggttag
tcaataactc aaattctctg tctatgacaa ctgctgtatg 4860actatatgaa gaaatgggat
aaagatgcta tagtcactcg ag 4902920836DNAartificial
sequencesynthetic polynucleotide (transgenic DNA construct + 5' and
3'arms) 9agcgcgcgta atacgactca ctatagggcg aattggagct ccaccgcggt
ggcggccgct 60cctgagagaa cagactctgg aaatagatgg gacttaagga gctaagatct
agagctcatc 120tacagagcag aatcccagcc aagagaacaa agaatactgg ctctctctcc
tgttccctac 180tcctagagtt ctaaaacaca ctatagggaa gggagcctct agacctccgt
ccattcccca 240tcttgctcat tccatcttcc catgtcccca ggtctccaag ccacagacac
tacctttcct 300attcacccac ctttctgtgt ccctaggtcc ccaggccata gtcacctccc
cccacacaca 360ccccactcac cctgccccat ctatgcccct agatgcttac ttaccagagt
cttttgtctg 420acgtggggct acaagcatct atgctcccta agcacctact gctgacctgt
aggacccagc 480tctgaaccaa ctcatataag taaatacaga ctctcccctg tcttaggatg
gcctcctgga 540tcaggaggag accactgcca aagaaccttc tctcagagca ctgaactcct
cccctgtacc 600acttaggaca gacctgagac ctattattac tgattaccag agctctggca
gtgaccacgg 660aggagatagg tccaccctgg acacaggaaa cacagcagca gagatactgc
tccatcacaa 720cagtagagtg acactttaga ctttaatttg ggtcactttc ctgctgcaga
ggtgggatca 780gaaagcaaag agcagtatga gtgcctgata ggcacccaag tacactatag
agtactcatg 840gtgaataagg tacctccatg gcttcccagg gaggggcagc cccaccccca
ccatcacaga 900cctttctcca tagttgataa ctcagacaca agtgaatgac agatggacct
ccatctactc 960ttattttaaa aagaagacaa accccacagg ctcgagaact ttagcgactg
ttttgagaga 1020aatcattggt ccctgactca agagatgact ggcagattgg ggatcagaat
acccatactc 1080tgtggctagt gtgaggttta agcctcagag tccctgtggt ctctgactgg
tgcaaggttt 1140tgactaagcg gagcaccaca gtgctaactg ggaccacggt gacacgtggc
tcaacaaaaa 1200ccttctgttt ggagctctcc aggggcagcc tgagctatga ggaagtagag
aggcttgaga 1260aatctgagga agaaaagagt agatctgaga ggaaaggtag ctttctggag
gtcaggagac 1320agtgcagaga agaacgagtt actgtggaca ggtcttagat ggggaaagaa
tgagcaaatg 1380caagcatcag aagggtggat gcaatgtcct gccaaggact taccaagagg
atccccggac 1440agagcaggca ggtggagttg actgagagga cagggtaggt gcaggtccct
ctctcgtttc 1500ctttctcctt ctcctgtttc cttcctctct tgtcacaggt ctcactatgc
tagccaaggc 1560tagcctgaaa gattaccatc ctacagatgg gcccatccag ttgagttaag
gtggagatct 1620ctccaaacat ctgagtttct gaggcttgga tgccactggg gacgccaagg
gactttgggc 1680tgggtttggt tggccccaga tgaagggcta cttcactggg tctataatta
ctctgatgtc 1740taggaccagg gggctcaggt cactcaggtc aggtgagtcc tgcatctggg
gactgtgggg 1800ttcaggtgtc ctaaggcagg atgtggagag agttttagta taggaacaga
ggcagaacag 1860agactgtgct actggtactt cgatgtctgg ggcgcaggga ccacggtcac
cgtctcctca 1920ggtaagctgg cttttttctt tctgcacatt ccattctgaa atgggaaaag
atattctcag 1980atctccccat gtcaggccat ctgccacact ctgcatgctg cagaagcttt
tctgtaagga 2040tagggtcttc actcccagga aaagaggcag tcagaggcta gctgcctgtg
gaacagtgac 2100aatcatggaa aataggcatt tacattgtta ggctacatgg gtagatgggt
ttttgtacac 2160ccactaaagg ggtctatgat agtgtgacta ctttgactac tggggccaag
gcaccactct 2220cacagtctcc tcaggtgagt ccttacaacc tctctcttct attcagctta
aatagatttt 2280actgcatttg ttggggggga aatgtgtgta tctgaatttc aggtcatgaa
ggactaggga 2340caccttggga gtcagaaagg gtcattggga gccctggctg acgcagacag
acatcctcag 2400ctcccatact tcatggccag agatttatag ggatcctggc cagcattgcc
gctaggtccc 2460tctcttctat gctttctttg tccctcactg gcctccatct gagataatcc
tggagcccta 2520gccaaggatc atttattgtc aggggtctaa tcattgttgt cacaatgtgc
ctggtttgct 2580tactggggcc aagggactct ggtcactgtc tctgcaggtg agtcctaact
tctcccattc 2640taaatgcatg ttggggggat tctgagcctt caggaccaag attctctgca
aacgggaatc 2700aagattcaac ccctttgtcc caaagttgag acatgggtct gggtcaggga
ctctctgcct 2760gctggtctgt ggtgacatta gaactgaagt atgatgaagg atctgccaga
actgaagctt 2820gaagtctgag gcagaatctt gtccagggtc tatcggactc ttgtgagaat
taggggctga 2880cagttgatgg tgacaatttc agggtcagtg actgtctggt ttctctgagg
tgaggctgga 2940atataggtca ccttgaagac ttaagagggg tccagggggc ttctgcacag
gcagggaaca 3000gaatgtggaa caatgacttg aatggttgat tcttgtgtga caccaggaat
tggcataatg 3060tctgagttgc ccaggggtga ttctagtcag actctggggt ttttgtcggg
tatagaggaa 3120aaatccacta ttgtgattac tatgctatgg actactgggg tcaaggaacc
tcagtcaccg 3180tctcctcagg taagaatggc ctctccaggt ctttattttt aacctttgtt
atggagtttt 3240ctgagcattg cagactaatc ttggatattt gtccctgagg gagccggctg
agagaagttg 3300ggaaataaac tgtctaggga tctcagagcc tttaggacag attatctcca
catctttgaa 3360aaactaagaa tctgtgtgat ggtgttggtg gagtccctgg atgatgggat
agggactttg 3420gaggctcatt tgagggagat gctaaaacaa tcctatggct ggagggatag
ttggggctgt 3480agttggagat tttcagtttt tagaataaaa gtattagctg cggaatatac
ttcaggacca 3540cctctgtgac agcatttata cagtatccga tgcataggga caaagagtgg
agtggggcac 3600tttctttaga tttgtgagga atgttccaca ctagattgtt taaaacttca
tttgttggaa 3660ggagagctgt cttagtgatt gagtcaaggg agaaaggcat ctagcctcgg
tctcaaaagg 3720gtagttgctg tctagagagg tctggtggag cctgcaaaag tccagctttc
aaaggaacac 3780agaagtatgt gtatggaata ttagaagatg ttgcttttac tcttaagttg
gttcctagga 3840aaaatagtta aatactgtga ctttaaaatg tgagagggtt ttcaagtact
cattttttta 3900aatgtccaaa atttttgtca atcaatttga ggtcttgttt gtgtagaact
gacattactt 3960aaagtttaac cgaggaatgg gagtgaggct ctctcataac ctattcagaa
ctgactttta 4020acaataataa attaagtttc aaatattttt aaatgaattg agcaatgttg
agttggagtc 4080aagatggccg atcagaacca gaacacctgc agcagctggc aggaagcagg
tcatgtggca 4140aggctatttg gggaagggaa aataaaacca ctaggtaaac ttgtagctgt
ggtttgaaga 4200agtggttttg aaacactctg tccagcccca ccaaaccgaa agtccaggct
gagcaaaaca 4260ccacctgggt aatttgcatt tctaaaataa gttgaggatt cagccgaaac
tggagaggtc 4320ctcttttaac ttattgagtt caacctttta attttagctt gagtagttct
agtttcccca 4380aacttaagtt tatcgacttc taaaatgtat ttagaattca ttttcaaaat
taggttatgt 4440aagaaattga aggactttag tgtctttaat ttctaatata tttagaaaac
ttcttaaaat 4500tactctatta ttcttccctc tgattattgg tctccattca attcttttcc
aatacccgaa 4560gcatttacag tgactttgtt catgatcttt tttagttgtt tgttttgcct
tactattaag 4620actttgacat tctggtcaaa acggcttcac aaatcttttt caagaccact
ttctgagtat 4680tcattttagg agaaagactt tttttttaaa tgaatgcaat tatctagact
tatttcagtt 4740gaacatgctg gttggtggtt gagaggacac tcagtcagtc agtggcgtga
agggcttcta 4800agccagtcca catgctctgt gtgaactccc tctggccctg cttattgttg
aatgggccaa 4860aggtctgaga ccaggctgct gctgggtagg cctggacttt gggtctccca
cccagacctg 4920ggaatgtatg gttgtggctt ctgccaccca tccacctggc tgctcatgga
ccagccagcc 4980tcggtggctt tgaaggaaca attccacaca aagactctgg acctctccga
aaccaggcac 5040cgcaaatggt aagccagagg cagccacagc tgtggctgct gctcttaaag
cttgtaaact 5100gtttctgctt aagagggact gagtcttcag tcattgcttt agggggagaa
agagacattt 5160gtgtgtcttt tgagtaccgt tgtctgggtc actcacattt aactttcctt
gaaaaactag 5220actcgacatc gattgtcgag gaattccgat catattcaat aacccttaat
ataacttcgt 5280ataatgtatg ctatacgaag ttattaggtc tgaagaggag tttacgtcca
gccttcgaag 5340ggtcctcagg gagtgcatcc gccccaaccc ttttccccct cgtctcctgt
gagaattccc 5400cgtcggatac gagcagcgtg gccgttggct gcctcgcaca ggacttcctt
cccgactcca 5460tcactttctc ctggaaatac aagaacaact ctgacatcag cagcacccgg
ggcttcccat 5520cagtcctgag agggggcaag tacgcagcca cctcacaggt gctgctgcct
tccaaggacg 5580tcatgcaggg cacagacgaa cacgtggtgt gcaaagtcca gcaccccaac
ggcaacaaag 5640aaaagaacgt gcctcttcca ggtgagggcc gggcccagcc accgggacag
agagggagcc 5700gaaggggggc gggagtggcg ggcaccgggc tgacacgtgt ccctcactgc
agtgattgct 5760gagctgcctc ccaaagtgag cgtcttcgtc ccaccccgcg acggcttctt
cggcaacccc 5820cgcaagtcca agctcatctg ccaggccacg ggtttcagtc cccggcagat
tcaggtgtcc 5880tggctgcgcg aggggaagca ggtggggtct ggcgtcacca cggaccaggt
gcaggctgag 5940gccaaagagt ctgggcccac gacctacaag gtgaccagca cactgaccat
caaagagagc 6000gactggctca gccagagcat gttcacctgc cgcgtggatc acaggggcct
gaccttccag 6060cagaatgcgt cctccatgtg tgtccccggt gagtgacctg tccccagggg
cagcacccac 6120cgacacacag gggtccactc gggtctggca ttcgccaccc cggatgcagc
catctactcc 6180ctgagccttg gcttcccaga gcggccaagg gcaggggctc gggcggcagg
acccctgggc 6240tcggcagagg cagttgctac tctttgggtg ggaaccatgc ctccgcccac
atccacacct 6300gccccacctc tgactccctt ctcttgactc cagatcaaga cacagccatc
cgggtcttcg 6360ccatcccccc atcctttgcc agcatcttcc tcaccaagtc caccaagttg
acctgcctgg 6420tcacagacct gaccacctat gacagcgtga ccatctcctg gacccgccag
aatggcgaag 6480ctgtgaaaac ccacaccaac atctccgaga gccaccccaa tgccactttc
agcgccgtgg 6540gtgaggccag catctgcgag gatgactgga attccgggga gaggttcacg
tgcaccgtga 6600cccacacaga cctgccctcg ccactgaagc agaccatctc ccggcccaag
ggtaggcccc 6660actcttgccc ctcttctgca ctcctgcaac tccttgcctc tgggggcatg
gtggaaagca 6720cccctcactc ccccgttgtc tgggcaactg gggaaaaggg gactcaaccc
cagcccacag 6780gctggtcccc ccactgcccc gccctcacca ccatctctgt tcacaggggt
ggccctgcac 6840aggcccgatg tctacttgct gccaccagcc cgggagcagc tgaacctgcg
ggagtcggcc 6900accatcacgt gcctggtgac gggcttctct cccgcggacg tcttcgtgca
gtggatgcag 6960agggggcagc ccttgtcccc ggagaagtat gtgaccagcg ccccaatgcc
tgagccccag 7020gccccaggcc ggtacttcgc ccacagcatc ctgaccgtgt ccgaagagga
atggaacacg 7080ggggagacct acacctgcgt ggtggcccat gaggccctgc ccaacagggt
caccgagagg 7140accgtggaca agtccaccgg taaacccacc ctgtacaacg tgtccctggt
catgtccgac 7200acagctggca cctgctactg accctgctgg cctgcccaca ggctcggggc
ggctggccgc 7260tctgtgtgtg catgcaaact aaccgtgtca acggggtgag atgttgcatc
ttataaaatt 7320agaaataaaa agatccattc aaaagatact ggtcctgagt gcacgatgct
ctggcctact 7380ggggcggcgg ctgtgctgca cccaccctgc gcctcccctg cagaacacct
tcctccacag 7440cccccacccc tgcctcaccc acctgcgtgc ctcagtggct tctagaaacc
cctgaattcc 7500ctgcagctgc tcacagcagg ctgacctcag acttgccatt cctcctactg
cttccagaaa 7560gaaagctgaa agcaaggcca cacgtataca ggcagcacac aggcatgtgt
ggatacacat 7620ggacagacac ggacacacac aaacacatgg acacacagag acgtgctaac
ccatgggcac 7680acacatacac agacatggac ccacacacaa acatatgtgg acacacatgt
acaaacatgc 7740acaggcacac aaagagaaca ctgactacag gcacacacac acacgggcac
acacatggat 7800atgtgcacac atggacacat acatgtgcag gacatgcaca cacacagaca
cactagcaca 7860gaggcataca cacacagaca cacacattca caaacacaca tgtgcatgca
aacacacaca 7920catgtacaga cacgagtaca tggacacatg cacacccaga gacacactga
cacagacaca 7980caggagcatg tgatacacta acacgtggac acacacgtct acccacaggc
acacaacaga 8040tggacacgcg tacacagaca tgcacacacc cacaggcaca acacgtgcgc
atgccggccg 8100gcccccgccc acattctccc agggccctgc cggatactct gtccctgcag
cagtttgctc 8160cctgcgctgt gctggccccg gggctttggg cccaggctct gcttgtcctt
ctgtctctgc 8220ttggaggtgc tgccatggca cccagcttgg gctctgcctg gggagcggag
gccccaggga 8280tagcatgtga cccctgctga ggccaggctc ctgatgaagg cagcagatag
cccccacacc 8340caccggtgag cagaaccaga gcctgtgcca tgtgctgaga gcaggcagtg
actaagcata 8400tgggcccaga gggcagagtg gctgccctgg gcagctgctc ctcttagcgg
gaggcctcag 8460gagatgagct agagcaagtc tgcccctgca aataccacct gctccccaac
ccacagcagg 8520gagcaggcga ggtcagacag cagcagcccg ggaaggaccg agccccagca
gggaaggcag 8580ggcccgagtg aggtctccac acccaacgca cagtgctgtc tctaactggg
gccacctccg 8640agtccccgcc acactcttgg ccctttggag tcctgggctc caggtgtctc
ccaagggccc 8700atctgtgcag gggatgcaac cccccgaatg tcctcatccc actgtggagc
tcaggtctct 8760gtctgctccc tgggtcctgg cagggtagga caagtccgcc aggatgtccc
catgcagact 8820ctgctccaag agggagctgg agagtcaggg ccttggtgag ggagtcagga
tcgggttccc 8880cccagctcag tcctcccacc tgccagcccc cacagcacag ggcagggcca
caccccctgc 8940ttccccctcc aggagagtca ggacatgctg gccgctgctc cgctggggcc
ccgccctcca 9000gcccccacct tggtctgtgt gctgcatccc ccacgctctc tctgccaccc
caggactctg 9060aggaaaagac ctcagagtcc cagccctgcc cagtctcggc ctgtgccccc
gctgcatcag 9120gctttcaggg gcccagccca tgccctgggc agtgcccgag cccccctgca
cttgctctcc 9180ccacccctgg gtgcagcaca gcctaggggc caagggtggg cctagaggat
gggccccggg 9240ggggctttgc tgggtgccac cccagcctga ccctattccc ccgtgctgtg
tctcctgcag 9300agggggaggt gagcgccgac gaggagggct ttgagaacct gtgggccacc
gcctccacct 9360tcatcgtcct cttcctcctg agcctcttct acagtaccac cgtcaccttg
ttcaaggtag 9420cacggctgtg gcacagggag gagggtgcag ggcgagtgtg gggcccaggg
agcagcctgg 9480gctggacgtc tagcccggag gcccccacac caccccactg ggtcatctct
gccccggctc 9540ccttcccgac cacggggaaa gcatttcaca ctgtctctgt tgcctgtagg
tgaaatgatc 9600ccaacagaag aacatcggag accagagaga ggaactcaaa ggggcgctgc
ctccgggtct 9660ggggtcctgg cctgcgtggc ctgttggcac gtgtttctct tccccgcccg
gcctccagtt 9720gtgtgctctc acacaggctt ctttctcgac cggcaggggc tggctggctt
gcaggccacg 9780aggtggggct ctaccccaca ctgctttgct gtgtatacgc ttgttgcctg
aaataaatat 9840gcacatttta tccatgaaac tgctttctgg tgagggtttg tttctttttc
aaaactttcc 9900tgctacaggg cattcaagcc atcgatgtcg aggaattccg atcatattca
ataaccctta 9960atataacttc gtataatgta tgctatacga agttattagg tctgaagagg
agtttacgtc 10020cagccaagct agcttggctg caggtcgagc agtgtggttt tcaagaggaa
gcaaaaagcc 10080tctccaccca ggcctggaat gtttccaccc aatgtcgagc agtgtggttt
tgcaagagga 10140agcaaaaagc ctctccaccc aggcctggaa tgtttccacc caatgtcgag
cagtgtggtt 10200ttgcaagagg aagcaaaaag cctctccacc caggcctgga atgtttccac
ccaatgtcga 10260gcaaaccccg cccagcgtct tgtcattggc gaattcgaac acgcagatgc
agtcggggcg 10320gcgcggtccc caggtccact tcgcatatta aggtgacgcg tgtggcctcg
aacaccgagc 10380gaccctgcag ccaatatggg atcggccatt gaacaagatg gattgcacgc
aggttctccg 10440gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
cggctgctct 10500gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
caagaccgac 10560ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
gctggccacg 10620acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
ggactggctg 10680ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
tgccgagaaa 10740gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
tacctgccca 10800ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
agccggtctt 10860gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
actgttcgcc 10920aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
cgatgcctgc 10980ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
tggccggctg 11040ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
tgaagagctt 11100ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
cgattcgcag 11160cgcatcgcct tctatcgcct tcttgacgag ttcttctgag gggatcggca
ataaaaagac 11220agaataaaac gcacgggtgt tgggtcgttt gttcggatca gcttccgatc
atattcaata 11280acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtct
gaagaggagt 11340ttacgtccag ccaagctaac ttggcgcccc cactagggtc gaggagcttg
gtaccgagct 11400cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttgatgaa
tgagcctggc 11460atctggctcc ctgccacggg gtccccagct cccccatcca ggccccccag
gcctgatggg 11520cgctggcctg aggctggcac tgactaggtt ctgtcctcac agcctccaca
cagagcccat 11580ccgtcttccc cttgacccgc tgctgcaaaa acattccctc caatgccacc
tccgtgactc 11640tgggctgcct ggccacgggc tacttcccgg agccggtgat ggtgacctgg
gacacaggct 11700ccctcaacgg gacaactatg accttaccag ccaccaccct cacgctctct
ggtcactatg 11760ccaccatcag cttgctgacc gtctcgggtg cgtgggccaa gcagatgttc
acctgccgtg 11820tggcacacac tccatcgtcc acagactggg tcgacaacaa aaccttcagc
ggtaagagag 11880ggccaagctc agagaccaca gttcccagga gtgccaggct gagggctggc
agagtgggca 11940ggggttgagg gggtgggtgg gctcaaacgt gggaacaccc agcatgcctg
gggacccggg 12000ccaggacgtg ggggcaagag gagggcacac agagctcaga gagcccaaca
accctcatga 12060ccaccagctc tcccccagtc tgctccaggg acttcacccc gcccaccgtg
aagatcttac 12120agtcgtcctg cgacggcggc gggcacttcc ccccgaccat ccagctcctg
tgcctcgtct 12180ctgggtacac cccagggact atcaacatca cctggctgga ggacgggcag
gtcatggacg 12240tggacttgtc caccgcctct accacgcagg agggtgagct ggcctccaca
caaagcgagc 12300tcaccctcag ccagaagcac tggctgtcag accgcaccta cacctgccag
gtcacctatc 12360aaggtcacac ctttgaggac agcaccaaga agtgtgcagg tacgttccca
cctgccctgg 12420tggccgccac ggaggccaga gaagaggggc gggtgggcct cacacagccc
tccggtgtac 12480cacagattcc aacccgagag gggtgagcgc ctacctaagc cggcccagcc
cgttcgacct 12540gttcatccgc aagtcgccca cgatcacctg tctggtggtg gacctggcac
ccagcaaggg 12600gaccgtgaac ctgacctggt cccgggccag tgggaagcct gtgaaccact
ccaccagaaa 12660ggaggagaag cagcgcaatg gcacgttaac cgtcacgtcc accctgccgg
tgggcacccg 12720agactggatc gagggggaga cctaccaatg cagggtgacc cacccccacc
tgcccagggc 12780cctcatgcgg tccacgacca agaccagcgg tgagccatgg gcaggccggg
gtcgtggggg 12840aagggaggga gcgagtgagc ggggcccggg ctgaccccac gtctggccac
aggcccgcgt 12900gctgccccgg aagtctatgc gtttgcgacg ccggagtggc cggggagccg
ggacaagcgc 12960accctcgcct gcctgatcca gaacttcatg cctgaggaca tctcggtgca
gtggctgcac 13020aacgaggtgc agctcccgga cgcccggcac agcacgacgc agccccgcaa
gaccaagggc 13080tccggcttct tcgtcttcag ccgcctggag gtgaccaggg ccgaatggga
gcagaaagat 13140gagttcatct gccgtgcagt ccatgaggca gcgagcccct cacagaccgt
ccagcgagcg 13200gtgtctgtaa atcccggtaa atgacgtact cctgcctccc tccctcccag
ggctccatcc 13260agctgtgcag tggggaggac tggccagacc ttctgtccac tgttgcaatg
accccaggaa 13320gctaccccca ataaactgtg cctgctcaga gccccaggta cacccattct
tgggagcggg 13380cagggctgtg ggcaggtgca tcttggcaca gaggaatggg ccccccagga
ggggcagtgg 13440gaggaggtgg gcagggctga gtccccccag gagaggtggt gggaggaggt
gggcaggggt 13500gaggtgccac tcatccatct gccttcgtgt cagggttatt tgtcaaacag
catatctgca 13560gggactcatc acagctaccc cgggcctctc tgcccccact ctgggtctac
cccctccaag 13620gagtccaaag acccagggga ggtcctcagg gaaggggcaa gggagccccc
acagccctcc 13680ctcttggggg cttggcttct acccccctgg acaggagccc ctgcaccccc
aggtatagat 13740gggcacacag gcccctccag gtggaaaaac agccctaagt gaaaccccca
cacagacaca 13800cacaacccga cagccctcgc ccaagtctgt gccactggcg ttcgcctctc
tgccctgtcc 13860cgccttgccg agtcctggcc ccagcaccgg ggccggtgga gccgagccca
ctcacacccc 13920gcagcctccg ccaccctgcc ctgtgggcac accaggccca ggtcagagcc
aggccccctc 13980tcctactgcc ccccaccgcc ccttggtcca tcctgaatcg gcctccaggg
gatcgccagc 14040ctcacacacc cggtctcgcc cactcacgcc tcactcaagg cacagctgtg
cacacactag 14100gccccatagc aactccacag caccctgtac caccaccagg gcgccataga
caccccacac 14160gtggtcacac gtggcccaca ctccgcctct cacgctgcct ccagccaggc
tactgccaag 14220cccttcctct gagccatacc tgggccgctg gatcccagag agaaatggag
aggccctcac 14280gtggtgtcct ccagtccaac cctccctgtc accctgtcag cagcacccca
cagccaaaca 14340caggatggat gcgtgggctc catcccccac tcacccacac ctgaacccca
gagcaggcta 14400cgtgcccctc acagacctca aacccacatg tgcatctgac accccagatc
caaacgctcc 14460ccccggtcat gcacaccaag ggcacagcac ccaccaaatc cacacggaaa
cacgggcacc 14520gggcacccca tgagcacaaa gcccctccat gtctgaagac agtccctgca
caccgtcaca 14580gccatacatt cagcttcact ctcacgtccc agcccacctg cacccagctc
tgggcctgga 14640gcagcagaaa gaggtgtgag ggcccgaggc cggacctgca cctgctgatg
acccgggacc 14700agcaggcagc tcacggtgtt ggggaaggga gtggagggca cccagggcag
gagccagagg 14760gaccaggctg gtgggcgggg ccgggccggg gtagggccag gaggcagctc
tggacaccca 14820caggcctggg ctcatagtcc acaccaggac agcccctcag agcacccatg
cagtgagtcc 14880caggtcttgg gagccaggcc gcagagctca cgcatccttc cgagggccct
gagtgaggcg 14940gccactgctg cgccgagggg ttgggtcctt ctctggggag ggcgtggggt
ctagagaggc 15000ggagtggagg taaccagagg tcaggagaga agccgtaagg aacagaggga
aaatggggcc 15060agagtcgggg cgcagggacg agaggtcagg agtggtcggc ctggccctgg
gccgttgact 15120gactcgggac ctgggtgccc accctcaggg ctggctggcg gctccgcgca
gtcccagagg 15180gccccggata gggtgctctg ccactccgga cagcagcagg gactgccgag
agcggcagga 15240ggctctgtcc cccacccccg ctgccactgt ggagccggga gggctgactg
gccaggtccc 15300ccagagctgg acgtgtgcgt ggaggaggcc gagggcgagg cgccgtggac
gtggaccggc 15360ctctgcatct tcgccgcact cttcctgctc agcgtgagct acagcgccgc
cctcacgctc 15420ctcatggtgg gcacccacct ccaggggccc agccagggca gggggttggg
cagagccagc 15480agagcgccct gacccacgcc ctcccctcag gtgcagcggt tcctctcagc
cacgcggcag 15540gggaggcccc agacctccct cgactacacc aacgtcctcc agccccacgc
ctaggccgcg 15600ggccactcac gctccaccag gcccagcttt ttctctgcca gcgcctgagc
ctccctcggg 15660ctgcaccctg ccctgggtgg gaaaagggaa gcagacaaga aaagggggca
caaggtcact 15720actgtgggct gatggccagt gaacctgagc ccagaggggc cggctcagcc
gcaaggttac 15780aggcgccgag agaaccacca gtcgcagccc ccacccgaaa accgtgtctg
tcccttcaac 15840agagtcatcg aggaggggtg gctgctagcc gttctgagct catcgaaggg
cgaattctgc 15900agatatccat cacactggcg gccgctcgac ctaggatctc ctgtctgaca
ggaggcaaga 15960agacagattc ttacccctcc atttctcttt tatccctctc tggtcctcag
agagtcagtc 16020cttcccaaat gtcttccccc tcgtctcctg cgagagcccc ctgtctgata
agaatctggt 16080ggccatgggc tgcctggccc gggacttcct gcccagcacc atttccttca
cctggaacta 16140ccagaacaac actgaagtca tccagggtat cagaaccttc ccaacactga
ggacaggggg 16200caagtaccta gccacctcgc aggtgttgct gtctcccaag agcatccttg
aaggttcaga 16260tgaatacctg gtatgcaaaa tccactacgg aggcaaaaac agagatctgc
atgtgcccat 16320tccaggtaag aaccaaaccc tcccagcagg ggtgcccagg cccaggcatg
gcccagaggg 16380agcagcgggt ggggcttagg ccaagctgag ctcacacctt gacctttcat
tccagctgtc 16440gcagagatga accccaatgt gttcgtccca ccacgggatg gcttctctgg
ccctgcacca 16500cgcaagtcta aactcatctg cgaggccacg aacttcactc caaaaccgat
cacagtatcc 16560tggctaaagg atgggaagct cgtggaatct ggcttcacca cagatccggt
gaccatcgag 16620aacaaaggat ccacacccca aacctacaag gtcataagca cacttaccat
ctctgaaatc 16680gactggctga acctgaatgt gtacacctgc cgtgtggatc acaggggtct
caccttcttg 16740aagaacgtgt cctccacatg tgctgccagt gagtggcctg ggataagccc
aatgcctagc 16800cctcccagat tagggaagtc ctcctacaat tatggccaat gccacccaga
catggtcatt 16860tgctccttga actttggctc cccagagtgg ccaaggacaa gaatgagcaa
taggcagtag 16920aggggtgaga atcagctgga aggaccagca tcttccctta agtaggtttg
ggggatggag 16980actaagcttt tttccaactt cacaactaga tatgtcataa cctgacacag
tgttctcttg 17040actgcaggtc cctccacaga catcctaacc ttcaccatcc ccccctcctt
tgccgacatc 17100ttcctcagca agtccgctaa cctgacctgt ctggtctcaa acctggcaac
ctatgaaacc 17160ctgaatatct cctgggcttc tcaaagtggt gaaccactgg aaaccaaaat
taaaatcatg 17220gaaagccatc ccaatggcac cttcagtgct aagggtgtgg ctagtgtttg
tgtggaagac 17280tggaataaca ggaaggaatt tgtgtgtact gtgactcaca gggatctgcc
ttcaccacag 17340aagaaattca tctcaaaacc caatggtagg tatcccccct tcccttcccc
tccaattgca 17400ggacccttcc tgtacctcat agggagggca ggtcctcttc caccctatcc
tcactactgt 17460cttcatttac agaggtgcac aaacatccac ctgctgtgta cctgctgcca
ccagctcgtg 17520agcaactgaa cctgagggag tcagccacag tcacctgcct ggtgaagggc
ttctctcctg 17580cagacatcag tgtgcagtgg cttcagagag ggcaactctt gccccaagag
aagtatgtga 17640ccagtgcccc gatgccagag cctggggccc caggcttcta ctttacccac
agcatcctga 17700ctgtgacaga ggaggaatgg aactccggag agacctatac ctgtgttgta
ggccacgagg 17760ccctgccaca cctggtgacc gagaggaccg tggacaagtc cactggtaaa
cccacactgt 17820acaatgtctc cctgatcatg tctgacacag gcggcacctg ctattgacca
tgctagcgct 17880caaccaggca ggccctgggt gtccagttgc tctgtgtatg caaactaacc
atgtcagagt 17940gagatgttgc attttataaa aattagaaat aaaaaaaatc cattcaaacg
tcactggttt 18000tgattataca atgctcatgc ctgctgagac agttgtgttt tgcttgctct
gcacacaccc 18060tgcatacttg cctccaccct ggcccttcct ctaccttgcc agtttcctcc
ttgtgtgtga 18120actcagtcag gcttacaaca gacagagtat gaacatgcga ttcctccagc
tacttcttga 18180tatatggctg aaagcttgcc taacctggtg caggcagcat tcaggcacat
atatagacac 18240acatgcattt atacatagat atataggtac acatgtgtag acacatacat
gaatgtgtat 18300tcatggacac acagacaaag gtacacatat atacacatga gttcatgcgc
acacacatgc 18360atggacactt acaaacgcct tcagagacaa ataggcatag acacacaacc
actcacagaa 18420acagatacca atatgcatgg tcctgtgtac acagaaacag actataggca
aatatacaca 18480aataaactat atagatacaa agatatgcat atacacacat gtacagaaac
atcttcacat 18540gtgtacacta acatgtggac aggtatagca cacagataca cctggactct
gaccagggct 18600gtaatctcca aggctcacgg ctcagagagc ctacactagg ctgggtcact
gatactcctc 18660aggagcccac tctatgattg ggagagataa ccccaggtac aaagtatgcc
tatctgtctc 18720aacaccatgg ggcagaagat actccactaa ccacccatga cagaaagtta
gccttggctg 18780tgtctccatt aatagaacac ctcagaagac caatgtgaaa ttgcctaacc
cactcacacc 18840caccctgatc tccagttcaa aatgcagaaa acataatgca gttgtccaaa
agatgcccca 18900accacacaca cacacacaca cacacacaca cacacacaca cacacacaca
cacacacaca 18960caccatcaag gagcctctgt aaggagtcac cacccaataa cactgcctct
ttgggctcat 19020atcctggaca ttcttcatat tcatatccat ttggggccta ggctttagat
atccccaagg 19080gctcatcttt acagggatca gagatcccaa taaatgccct ggtcccacag
cctccctcag 19140gtatctgtct gtttatctct tggtaccaag acccaacatt gctggcaggg
gtaggacaag 19200caacgcacgg gaactctgat caaagaaagt catgagatgc ctgagtcctt
caggaagtaa 19260ggagggacaa cctctggtat ccctgttctt attgctaaag cccaagagac
agggagacct 19320gctctaaatt ctcagtctaa acagcaccga tggcaccacc tgctcaggga
aagtccagag 19380cacaccaata tcattttgcc acagttcctg agtctgcctt tacccaggtc
catacattgc 19440atctgtcttg cttgctctgc tgccccaggg ctcctggaac aaaggctcca
aattagtgtg 19500tcctacagct tggcctgttc tgtgcctccg tctagcttga gctattaggg
gaccagtcaa 19560tactcgctaa gattctccag aaccatcagg gcaccccaac ccttatgcaa
atgctcagtc 19620accccaagac ttggcttgac cctccctctc tgtgtccctt catagagggg
gaggtgaatg 19680ctgaggagga aggctttgag aacctgtgga ccactgcctc caccttcatc
gtcctcttcc 19740tcctgagcct cttctacagc accaccgtca ccctgttcaa ggtagtatgg
ttgtggggct 19800gaggacacag ggctgggaca gggagtcacc agtcctcact gcctctacct
ctactcccta 19860caagtggaca gcaattcaca ctgtctctgt cacctgcagg tgaaatgact
ctcagcatgg 19920aaggacagca gagaccaaga gatcctccca cagggacact acctctgggc
ctgggatacc 19980tgactgtatg actagtaaac ttattcttac gtctttcctg tgttgccctc
cagcttttat 20040ctctgagatg gtcttctttc tagactgacc aaagactttt tgtcaacttg
tacaatctga 20100agcaatgtct ggcccacaga cagctgagct gtaaacaaat gtcacatgga
aataaatact 20160ttatcttgtg aactcacttt attgtgaagg aatttgtttt gtttttcaaa
cctttcctgc 20220ggtgttgaca gcccaaggat tatctgaata gagcttagga actggaaatg
gaacagtgca 20280gtctgatggt acttaaggga gaaagaggga aaggaggtgt ggaagaagaa
aaaagagaag 20340cagaggggga ggggagaagg gagagggaga gggagaggga gagggagagg
gagagggaga 20400gggagagaga gagagagaga gagagagaga gagagagcat gcactctaac
agcaaagtac 20460aacacaggca gccaatggat aacactctgg ttatctaccc tgatggaaga
agggaagtag 20520ggcagagaaa attccaggcc taatctccca aaagcaacag aacctggaaa
ctagcctcta 20580gccttaggtc tctgctctgt ccccagccca ccatcttggg ctggtgttgc
ttcaagctag 20640taatttaggt cttatcccaa agctttgtgg tatgtgggtg tgcctttggg
gagttggctg 20700agattttgaa gatgtttgta cctctcccac aacatgacaa gccctagggg
ttagtcaata 20760actcaaattc tctgtctatg acaactgctg tatgactata tgaagaaatg
ggataaagat 20820gctatagtca ctcgag
208361023669DNAartificial sequencesynthetic polynucleotide
(targeting vector) 10agcgcgcgta atacgactca ctatagggcg aattggagct
ccaccgcggt ggcggccgct 60cctgagagaa cagactctgg aaatagatgg gacttaagga
gctaagatct agagctcatc 120tacagagcag aatcccagcc aagagaacaa agaatactgg
ctctctctcc tgttccctac 180tcctagagtt ctaaaacaca ctatagggaa gggagcctct
agacctccgt ccattcccca 240tcttgctcat tccatcttcc catgtcccca ggtctccaag
ccacagacac tacctttcct 300attcacccac ctttctgtgt ccctaggtcc ccaggccata
gtcacctccc cccacacaca 360ccccactcac cctgccccat ctatgcccct agatgcttac
ttaccagagt cttttgtctg 420acgtggggct acaagcatct atgctcccta agcacctact
gctgacctgt aggacccagc 480tctgaaccaa ctcatataag taaatacaga ctctcccctg
tcttaggatg gcctcctgga 540tcaggaggag accactgcca aagaaccttc tctcagagca
ctgaactcct cccctgtacc 600acttaggaca gacctgagac ctattattac tgattaccag
agctctggca gtgaccacgg 660aggagatagg tccaccctgg acacaggaaa cacagcagca
gagatactgc tccatcacaa 720cagtagagtg acactttaga ctttaatttg ggtcactttc
ctgctgcaga ggtgggatca 780gaaagcaaag agcagtatga gtgcctgata ggcacccaag
tacactatag agtactcatg 840gtgaataagg tacctccatg gcttcccagg gaggggcagc
cccaccccca ccatcacaga 900cctttctcca tagttgataa ctcagacaca agtgaatgac
agatggacct ccatctactc 960ttattttaaa aagaagacaa accccacagg ctcgagaact
ttagcgactg ttttgagaga 1020aatcattggt ccctgactca agagatgact ggcagattgg
ggatcagaat acccatactc 1080tgtggctagt gtgaggttta agcctcagag tccctgtggt
ctctgactgg tgcaaggttt 1140tgactaagcg gagcaccaca gtgctaactg ggaccacggt
gacacgtggc tcaacaaaaa 1200ccttctgttt ggagctctcc aggggcagcc tgagctatga
ggaagtagag aggcttgaga 1260aatctgagga agaaaagagt agatctgaga ggaaaggtag
ctttctggag gtcaggagac 1320agtgcagaga agaacgagtt actgtggaca ggtcttagat
ggggaaagaa tgagcaaatg 1380caagcatcag aagggtggat gcaatgtcct gccaaggact
taccaagagg atccccggac 1440agagcaggca ggtggagttg actgagagga cagggtaggt
gcaggtccct ctctcgtttc 1500ctttctcctt ctcctgtttc cttcctctct tgtcacaggt
ctcactatgc tagccaaggc 1560tagcctgaaa gattaccatc ctacagatgg gcccatccag
ttgagttaag gtggagatct 1620ctccaaacat ctgagtttct gaggcttgga tgccactggg
gacgccaagg gactttgggc 1680tgggtttggt tggccccaga tgaagggcta cttcactggg
tctataatta ctctgatgtc 1740taggaccagg gggctcaggt cactcaggtc aggtgagtcc
tgcatctggg gactgtgggg 1800ttcaggtgtc ctaaggcagg atgtggagag agttttagta
taggaacaga ggcagaacag 1860agactgtgct actggtactt cgatgtctgg ggcgcaggga
ccacggtcac cgtctcctca 1920ggtaagctgg cttttttctt tctgcacatt ccattctgaa
atgggaaaag atattctcag 1980atctccccat gtcaggccat ctgccacact ctgcatgctg
cagaagcttt tctgtaagga 2040tagggtcttc actcccagga aaagaggcag tcagaggcta
gctgcctgtg gaacagtgac 2100aatcatggaa aataggcatt tacattgtta ggctacatgg
gtagatgggt ttttgtacac 2160ccactaaagg ggtctatgat agtgtgacta ctttgactac
tggggccaag gcaccactct 2220cacagtctcc tcaggtgagt ccttacaacc tctctcttct
attcagctta aatagatttt 2280actgcatttg ttggggggga aatgtgtgta tctgaatttc
aggtcatgaa ggactaggga 2340caccttggga gtcagaaagg gtcattggga gccctggctg
acgcagacag acatcctcag 2400ctcccatact tcatggccag agatttatag ggatcctggc
cagcattgcc gctaggtccc 2460tctcttctat gctttctttg tccctcactg gcctccatct
gagataatcc tggagcccta 2520gccaaggatc atttattgtc aggggtctaa tcattgttgt
cacaatgtgc ctggtttgct 2580tactggggcc aagggactct ggtcactgtc tctgcaggtg
agtcctaact tctcccattc 2640taaatgcatg ttggggggat tctgagcctt caggaccaag
attctctgca aacgggaatc 2700aagattcaac ccctttgtcc caaagttgag acatgggtct
gggtcaggga ctctctgcct 2760gctggtctgt ggtgacatta gaactgaagt atgatgaagg
atctgccaga actgaagctt 2820gaagtctgag gcagaatctt gtccagggtc tatcggactc
ttgtgagaat taggggctga 2880cagttgatgg tgacaatttc agggtcagtg actgtctggt
ttctctgagg tgaggctgga 2940atataggtca ccttgaagac ttaagagggg tccagggggc
ttctgcacag gcagggaaca 3000gaatgtggaa caatgacttg aatggttgat tcttgtgtga
caccaggaat tggcataatg 3060tctgagttgc ccaggggtga ttctagtcag actctggggt
ttttgtcggg tatagaggaa 3120aaatccacta ttgtgattac tatgctatgg actactgggg
tcaaggaacc tcagtcaccg 3180tctcctcagg taagaatggc ctctccaggt ctttattttt
aacctttgtt atggagtttt 3240ctgagcattg cagactaatc ttggatattt gtccctgagg
gagccggctg agagaagttg 3300ggaaataaac tgtctaggga tctcagagcc tttaggacag
attatctcca catctttgaa 3360aaactaagaa tctgtgtgat ggtgttggtg gagtccctgg
atgatgggat agggactttg 3420gaggctcatt tgagggagat gctaaaacaa tcctatggct
ggagggatag ttggggctgt 3480agttggagat tttcagtttt tagaataaaa gtattagctg
cggaatatac ttcaggacca 3540cctctgtgac agcatttata cagtatccga tgcataggga
caaagagtgg agtggggcac 3600tttctttaga tttgtgagga atgttccaca ctagattgtt
taaaacttca tttgttggaa 3660ggagagctgt cttagtgatt gagtcaaggg agaaaggcat
ctagcctcgg tctcaaaagg 3720gtagttgctg tctagagagg tctggtggag cctgcaaaag
tccagctttc aaaggaacac 3780agaagtatgt gtatggaata ttagaagatg ttgcttttac
tcttaagttg gttcctagga 3840aaaatagtta aatactgtga ctttaaaatg tgagagggtt
ttcaagtact cattttttta 3900aatgtccaaa atttttgtca atcaatttga ggtcttgttt
gtgtagaact gacattactt 3960aaagtttaac cgaggaatgg gagtgaggct ctctcataac
ctattcagaa ctgactttta 4020acaataataa attaagtttc aaatattttt aaatgaattg
agcaatgttg agttggagtc 4080aagatggccg atcagaacca gaacacctgc agcagctggc
aggaagcagg tcatgtggca 4140aggctatttg gggaagggaa aataaaacca ctaggtaaac
ttgtagctgt ggtttgaaga 4200agtggttttg aaacactctg tccagcccca ccaaaccgaa
agtccaggct gagcaaaaca 4260ccacctgggt aatttgcatt tctaaaataa gttgaggatt
cagccgaaac tggagaggtc 4320ctcttttaac ttattgagtt caacctttta attttagctt
gagtagttct agtttcccca 4380aacttaagtt tatcgacttc taaaatgtat ttagaattca
ttttcaaaat taggttatgt 4440aagaaattga aggactttag tgtctttaat ttctaatata
tttagaaaac ttcttaaaat 4500tactctatta ttcttccctc tgattattgg tctccattca
attcttttcc aatacccgaa 4560gcatttacag tgactttgtt catgatcttt tttagttgtt
tgttttgcct tactattaag 4620actttgacat tctggtcaaa acggcttcac aaatcttttt
caagaccact ttctgagtat 4680tcattttagg agaaagactt tttttttaaa tgaatgcaat
tatctagact tatttcagtt 4740gaacatgctg gttggtggtt gagaggacac tcagtcagtc
agtggcgtga agggcttcta 4800agccagtcca catgctctgt gtgaactccc tctggccctg
cttattgttg aatgggccaa 4860aggtctgaga ccaggctgct gctgggtagg cctggacttt
gggtctccca cccagacctg 4920ggaatgtatg gttgtggctt ctgccaccca tccacctggc
tgctcatgga ccagccagcc 4980tcggtggctt tgaaggaaca attccacaca aagactctgg
acctctccga aaccaggcac 5040cgcaaatggt aagccagagg cagccacagc tgtggctgct
gctcttaaag cttgtaaact 5100gtttctgctt aagagggact gagtcttcag tcattgcttt
agggggagaa agagacattt 5160gtgtgtcttt tgagtaccgt tgtctgggtc actcacattt
aactttcctt gaaaaactag 5220actcgacatc gattgtcgag gaattccgat catattcaat
aacccttaat ataacttcgt 5280ataatgtatg ctatacgaag ttattaggtc tgaagaggag
tttacgtcca gccttcgaag 5340ggtcctcagg gagtgcatcc gccccaaccc ttttccccct
cgtctcctgt gagaattccc 5400cgtcggatac gagcagcgtg gccgttggct gcctcgcaca
ggacttcctt cccgactcca 5460tcactttctc ctggaaatac aagaacaact ctgacatcag
cagcacccgg ggcttcccat 5520cagtcctgag agggggcaag tacgcagcca cctcacaggt
gctgctgcct tccaaggacg 5580tcatgcaggg cacagacgaa cacgtggtgt gcaaagtcca
gcaccccaac ggcaacaaag 5640aaaagaacgt gcctcttcca ggtgagggcc gggcccagcc
accgggacag agagggagcc 5700gaaggggggc gggagtggcg ggcaccgggc tgacacgtgt
ccctcactgc agtgattgct 5760gagctgcctc ccaaagtgag cgtcttcgtc ccaccccgcg
acggcttctt cggcaacccc 5820cgcaagtcca agctcatctg ccaggccacg ggtttcagtc
cccggcagat tcaggtgtcc 5880tggctgcgcg aggggaagca ggtggggtct ggcgtcacca
cggaccaggt gcaggctgag 5940gccaaagagt ctgggcccac gacctacaag gtgaccagca
cactgaccat caaagagagc 6000gactggctca gccagagcat gttcacctgc cgcgtggatc
acaggggcct gaccttccag 6060cagaatgcgt cctccatgtg tgtccccggt gagtgacctg
tccccagggg cagcacccac 6120cgacacacag gggtccactc gggtctggca ttcgccaccc
cggatgcagc catctactcc 6180ctgagccttg gcttcccaga gcggccaagg gcaggggctc
gggcggcagg acccctgggc 6240tcggcagagg cagttgctac tctttgggtg ggaaccatgc
ctccgcccac atccacacct 6300gccccacctc tgactccctt ctcttgactc cagatcaaga
cacagccatc cgggtcttcg 6360ccatcccccc atcctttgcc agcatcttcc tcaccaagtc
caccaagttg acctgcctgg 6420tcacagacct gaccacctat gacagcgtga ccatctcctg
gacccgccag aatggcgaag 6480ctgtgaaaac ccacaccaac atctccgaga gccaccccaa
tgccactttc agcgccgtgg 6540gtgaggccag catctgcgag gatgactgga attccgggga
gaggttcacg tgcaccgtga 6600cccacacaga cctgccctcg ccactgaagc agaccatctc
ccggcccaag ggtaggcccc 6660actcttgccc ctcttctgca ctcctgcaac tccttgcctc
tgggggcatg gtggaaagca 6720cccctcactc ccccgttgtc tgggcaactg gggaaaaggg
gactcaaccc cagcccacag 6780gctggtcccc ccactgcccc gccctcacca ccatctctgt
tcacaggggt ggccctgcac 6840aggcccgatg tctacttgct gccaccagcc cgggagcagc
tgaacctgcg ggagtcggcc 6900accatcacgt gcctggtgac gggcttctct cccgcggacg
tcttcgtgca gtggatgcag 6960agggggcagc ccttgtcccc ggagaagtat gtgaccagcg
ccccaatgcc tgagccccag 7020gccccaggcc ggtacttcgc ccacagcatc ctgaccgtgt
ccgaagagga atggaacacg 7080ggggagacct acacctgcgt ggtggcccat gaggccctgc
ccaacagggt caccgagagg 7140accgtggaca agtccaccgg taaacccacc ctgtacaacg
tgtccctggt catgtccgac 7200acagctggca cctgctactg accctgctgg cctgcccaca
ggctcggggc ggctggccgc 7260tctgtgtgtg catgcaaact aaccgtgtca acggggtgag
atgttgcatc ttataaaatt 7320agaaataaaa agatccattc aaaagatact ggtcctgagt
gcacgatgct ctggcctact 7380ggggcggcgg ctgtgctgca cccaccctgc gcctcccctg
cagaacacct tcctccacag 7440cccccacccc tgcctcaccc acctgcgtgc ctcagtggct
tctagaaacc cctgaattcc 7500ctgcagctgc tcacagcagg ctgacctcag acttgccatt
cctcctactg cttccagaaa 7560gaaagctgaa agcaaggcca cacgtataca ggcagcacac
aggcatgtgt ggatacacat 7620ggacagacac ggacacacac aaacacatgg acacacagag
acgtgctaac ccatgggcac 7680acacatacac agacatggac ccacacacaa acatatgtgg
acacacatgt acaaacatgc 7740acaggcacac aaagagaaca ctgactacag gcacacacac
acacgggcac acacatggat 7800atgtgcacac atggacacat acatgtgcag gacatgcaca
cacacagaca cactagcaca 7860gaggcataca cacacagaca cacacattca caaacacaca
tgtgcatgca aacacacaca 7920catgtacaga cacgagtaca tggacacatg cacacccaga
gacacactga cacagacaca 7980caggagcatg tgatacacta acacgtggac acacacgtct
acccacaggc acacaacaga 8040tggacacgcg tacacagaca tgcacacacc cacaggcaca
acacgtgcgc atgccggccg 8100gcccccgccc acattctccc agggccctgc cggatactct
gtccctgcag cagtttgctc 8160cctgcgctgt gctggccccg gggctttggg cccaggctct
gcttgtcctt ctgtctctgc 8220ttggaggtgc tgccatggca cccagcttgg gctctgcctg
gggagcggag gccccaggga 8280tagcatgtga cccctgctga ggccaggctc ctgatgaagg
cagcagatag cccccacacc 8340caccggtgag cagaaccaga gcctgtgcca tgtgctgaga
gcaggcagtg actaagcata 8400tgggcccaga gggcagagtg gctgccctgg gcagctgctc
ctcttagcgg gaggcctcag 8460gagatgagct agagcaagtc tgcccctgca aataccacct
gctccccaac ccacagcagg 8520gagcaggcga ggtcagacag cagcagcccg ggaaggaccg
agccccagca gggaaggcag 8580ggcccgagtg aggtctccac acccaacgca cagtgctgtc
tctaactggg gccacctccg 8640agtccccgcc acactcttgg ccctttggag tcctgggctc
caggtgtctc ccaagggccc 8700atctgtgcag gggatgcaac cccccgaatg tcctcatccc
actgtggagc tcaggtctct 8760gtctgctccc tgggtcctgg cagggtagga caagtccgcc
aggatgtccc catgcagact 8820ctgctccaag agggagctgg agagtcaggg ccttggtgag
ggagtcagga tcgggttccc 8880cccagctcag tcctcccacc tgccagcccc cacagcacag
ggcagggcca caccccctgc 8940ttccccctcc aggagagtca ggacatgctg gccgctgctc
cgctggggcc ccgccctcca 9000gcccccacct tggtctgtgt gctgcatccc ccacgctctc
tctgccaccc caggactctg 9060aggaaaagac ctcagagtcc cagccctgcc cagtctcggc
ctgtgccccc gctgcatcag 9120gctttcaggg gcccagccca tgccctgggc agtgcccgag
cccccctgca cttgctctcc 9180ccacccctgg gtgcagcaca gcctaggggc caagggtggg
cctagaggat gggccccggg 9240ggggctttgc tgggtgccac cccagcctga ccctattccc
ccgtgctgtg tctcctgcag 9300agggggaggt gagcgccgac gaggagggct ttgagaacct
gtgggccacc gcctccacct 9360tcatcgtcct cttcctcctg agcctcttct acagtaccac
cgtcaccttg ttcaaggtag 9420cacggctgtg gcacagggag gagggtgcag ggcgagtgtg
gggcccaggg agcagcctgg 9480gctggacgtc tagcccggag gcccccacac caccccactg
ggtcatctct gccccggctc 9540ccttcccgac cacggggaaa gcatttcaca ctgtctctgt
tgcctgtagg tgaaatgatc 9600ccaacagaag aacatcggag accagagaga ggaactcaaa
ggggcgctgc ctccgggtct 9660ggggtcctgg cctgcgtggc ctgttggcac gtgtttctct
tccccgcccg gcctccagtt 9720gtgtgctctc acacaggctt ctttctcgac cggcaggggc
tggctggctt gcaggccacg 9780aggtggggct ctaccccaca ctgctttgct gtgtatacgc
ttgttgcctg aaataaatat 9840gcacatttta tccatgaaac tgctttctgg tgagggtttg
tttctttttc aaaactttcc 9900tgctacaggg cattcaagcc atcgatgtcg aggaattccg
atcatattca ataaccctta 9960atataacttc gtataatgta tgctatacga agttattagg
tctgaagagg agtttacgtc 10020cagccaagct agcttggctg caggtcgagc agtgtggttt
tcaagaggaa gcaaaaagcc 10080tctccaccca ggcctggaat gtttccaccc aatgtcgagc
agtgtggttt tgcaagagga 10140agcaaaaagc ctctccaccc aggcctggaa tgtttccacc
caatgtcgag cagtgtggtt 10200ttgcaagagg aagcaaaaag cctctccacc caggcctgga
atgtttccac ccaatgtcga 10260gcaaaccccg cccagcgtct tgtcattggc gaattcgaac
acgcagatgc agtcggggcg 10320gcgcggtccc caggtccact tcgcatatta aggtgacgcg
tgtggcctcg aacaccgagc 10380gaccctgcag ccaatatggg atcggccatt gaacaagatg
gattgcacgc aggttctccg 10440gccgcttggg tggagaggct attcggctat gactgggcac
aacagacaat cggctgctct 10500gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
ttctttttgt caagaccgac 10560ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
ggctatcgtg gctggccacg 10620acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
aagcgggaag ggactggctg 10680ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
accttgctcc tgccgagaaa 10740gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
ttgatccggc tacctgccca 10800ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
ctcggatgga agccggtctt 10860gtcgatcagg atgatctgga cgaagagcat caggggctcg
cgccagccga actgttcgcc 10920aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
tgacccatgg cgatgcctgc 10980ttgccgaata tcatggtgga aaatggccgc ttttctggat
tcatcgactg tggccggctg 11040ggtgtggcgg accgctatca ggacatagcg ttggctaccc
gtgatattgc tgaagagctt 11100ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
tcgccgctcc cgattcgcag 11160cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
gggatcggca ataaaaagac 11220agaataaaac gcacgggtgt tgggtcgttt gttcggatca
gcttccgatc atattcaata 11280acccttaata taacttcgta taatgtatgc tatacgaagt
tattaggtct gaagaggagt 11340ttacgtccag ccaagctaac ttggcgcccc cactagggtc
gaggagcttg gtaccgagct 11400cggatccact agtaacggcc gccagtgtgc tggaattcgc
ccttgatgaa tgagcctggc 11460atctggctcc ctgccacggg gtccccagct cccccatcca
ggccccccag gcctgatggg 11520cgctggcctg aggctggcac tgactaggtt ctgtcctcac
agcctccaca cagagcccat 11580ccgtcttccc cttgacccgc tgctgcaaaa acattccctc
caatgccacc tccgtgactc 11640tgggctgcct ggccacgggc tacttcccgg agccggtgat
ggtgacctgg gacacaggct 11700ccctcaacgg gacaactatg accttaccag ccaccaccct
cacgctctct ggtcactatg 11760ccaccatcag cttgctgacc gtctcgggtg cgtgggccaa
gcagatgttc acctgccgtg 11820tggcacacac tccatcgtcc acagactggg tcgacaacaa
aaccttcagc ggtaagagag 11880ggccaagctc agagaccaca gttcccagga gtgccaggct
gagggctggc agagtgggca 11940ggggttgagg gggtgggtgg gctcaaacgt gggaacaccc
agcatgcctg gggacccggg 12000ccaggacgtg ggggcaagag gagggcacac agagctcaga
gagcccaaca accctcatga 12060ccaccagctc tcccccagtc tgctccaggg acttcacccc
gcccaccgtg aagatcttac 12120agtcgtcctg cgacggcggc gggcacttcc ccccgaccat
ccagctcctg tgcctcgtct 12180ctgggtacac cccagggact atcaacatca cctggctgga
ggacgggcag gtcatggacg 12240tggacttgtc caccgcctct accacgcagg agggtgagct
ggcctccaca caaagcgagc 12300tcaccctcag ccagaagcac tggctgtcag accgcaccta
cacctgccag gtcacctatc 12360aaggtcacac ctttgaggac agcaccaaga agtgtgcagg
tacgttccca cctgccctgg 12420tggccgccac ggaggccaga gaagaggggc gggtgggcct
cacacagccc tccggtgtac 12480cacagattcc aacccgagag gggtgagcgc ctacctaagc
cggcccagcc cgttcgacct 12540gttcatccgc aagtcgccca cgatcacctg tctggtggtg
gacctggcac ccagcaaggg 12600gaccgtgaac ctgacctggt cccgggccag tgggaagcct
gtgaaccact ccaccagaaa 12660ggaggagaag cagcgcaatg gcacgttaac cgtcacgtcc
accctgccgg tgggcacccg 12720agactggatc gagggggaga cctaccaatg cagggtgacc
cacccccacc tgcccagggc 12780cctcatgcgg tccacgacca agaccagcgg tgagccatgg
gcaggccggg gtcgtggggg 12840aagggaggga gcgagtgagc ggggcccggg ctgaccccac
gtctggccac aggcccgcgt 12900gctgccccgg aagtctatgc gtttgcgacg ccggagtggc
cggggagccg ggacaagcgc 12960accctcgcct gcctgatcca gaacttcatg cctgaggaca
tctcggtgca gtggctgcac 13020aacgaggtgc agctcccgga cgcccggcac agcacgacgc
agccccgcaa gaccaagggc 13080tccggcttct tcgtcttcag ccgcctggag gtgaccaggg
ccgaatggga gcagaaagat 13140gagttcatct gccgtgcagt ccatgaggca gcgagcccct
cacagaccgt ccagcgagcg 13200gtgtctgtaa atcccggtaa atgacgtact cctgcctccc
tccctcccag ggctccatcc 13260agctgtgcag tggggaggac tggccagacc ttctgtccac
tgttgcaatg accccaggaa 13320gctaccccca ataaactgtg cctgctcaga gccccaggta
cacccattct tgggagcggg 13380cagggctgtg ggcaggtgca tcttggcaca gaggaatggg
ccccccagga ggggcagtgg 13440gaggaggtgg gcagggctga gtccccccag gagaggtggt
gggaggaggt gggcaggggt 13500gaggtgccac tcatccatct gccttcgtgt cagggttatt
tgtcaaacag catatctgca 13560gggactcatc acagctaccc cgggcctctc tgcccccact
ctgggtctac cccctccaag 13620gagtccaaag acccagggga ggtcctcagg gaaggggcaa
gggagccccc acagccctcc 13680ctcttggggg cttggcttct acccccctgg acaggagccc
ctgcaccccc aggtatagat 13740gggcacacag gcccctccag gtggaaaaac agccctaagt
gaaaccccca cacagacaca 13800cacaacccga cagccctcgc ccaagtctgt gccactggcg
ttcgcctctc tgccctgtcc 13860cgccttgccg agtcctggcc ccagcaccgg ggccggtgga
gccgagccca ctcacacccc 13920gcagcctccg ccaccctgcc ctgtgggcac accaggccca
ggtcagagcc aggccccctc 13980tcctactgcc ccccaccgcc ccttggtcca tcctgaatcg
gcctccaggg gatcgccagc 14040ctcacacacc cggtctcgcc cactcacgcc tcactcaagg
cacagctgtg cacacactag 14100gccccatagc aactccacag caccctgtac caccaccagg
gcgccataga caccccacac 14160gtggtcacac gtggcccaca ctccgcctct cacgctgcct
ccagccaggc tactgccaag 14220cccttcctct gagccatacc tgggccgctg gatcccagag
agaaatggag aggccctcac 14280gtggtgtcct ccagtccaac cctccctgtc accctgtcag
cagcacccca cagccaaaca 14340caggatggat gcgtgggctc catcccccac tcacccacac
ctgaacccca gagcaggcta 14400cgtgcccctc acagacctca aacccacatg tgcatctgac
accccagatc caaacgctcc 14460ccccggtcat gcacaccaag ggcacagcac ccaccaaatc
cacacggaaa cacgggcacc 14520gggcacccca tgagcacaaa gcccctccat gtctgaagac
agtccctgca caccgtcaca 14580gccatacatt cagcttcact ctcacgtccc agcccacctg
cacccagctc tgggcctgga 14640gcagcagaaa gaggtgtgag ggcccgaggc cggacctgca
cctgctgatg acccgggacc 14700agcaggcagc tcacggtgtt ggggaaggga gtggagggca
cccagggcag gagccagagg 14760gaccaggctg gtgggcgggg ccgggccggg gtagggccag
gaggcagctc tggacaccca 14820caggcctggg ctcatagtcc acaccaggac agcccctcag
agcacccatg cagtgagtcc 14880caggtcttgg gagccaggcc gcagagctca cgcatccttc
cgagggccct gagtgaggcg 14940gccactgctg cgccgagggg ttgggtcctt ctctggggag
ggcgtggggt ctagagaggc 15000ggagtggagg taaccagagg tcaggagaga agccgtaagg
aacagaggga aaatggggcc 15060agagtcgggg cgcagggacg agaggtcagg agtggtcggc
ctggccctgg gccgttgact 15120gactcgggac ctgggtgccc accctcaggg ctggctggcg
gctccgcgca gtcccagagg 15180gccccggata gggtgctctg ccactccgga cagcagcagg
gactgccgag agcggcagga 15240ggctctgtcc cccacccccg ctgccactgt ggagccggga
gggctgactg gccaggtccc 15300ccagagctgg acgtgtgcgt ggaggaggcc gagggcgagg
cgccgtggac gtggaccggc 15360ctctgcatct tcgccgcact cttcctgctc agcgtgagct
acagcgccgc cctcacgctc 15420ctcatggtgg gcacccacct ccaggggccc agccagggca
gggggttggg cagagccagc 15480agagcgccct gacccacgcc ctcccctcag gtgcagcggt
tcctctcagc cacgcggcag 15540gggaggcccc agacctccct cgactacacc aacgtcctcc
agccccacgc ctaggccgcg 15600ggccactcac gctccaccag gcccagcttt ttctctgcca
gcgcctgagc ctccctcggg 15660ctgcaccctg ccctgggtgg gaaaagggaa gcagacaaga
aaagggggca caaggtcact 15720actgtgggct gatggccagt gaacctgagc ccagaggggc
cggctcagcc gcaaggttac 15780aggcgccgag agaaccacca gtcgcagccc ccacccgaaa
accgtgtctg tcccttcaac 15840agagtcatcg aggaggggtg gctgctagcc gttctgagct
catcgaaggg cgaattctgc 15900agatatccat cacactggcg gccgctcgac ctaggatctc
ctgtctgaca ggaggcaaga 15960agacagattc ttacccctcc atttctcttt tatccctctc
tggtcctcag agagtcagtc 16020cttcccaaat gtcttccccc tcgtctcctg cgagagcccc
ctgtctgata agaatctggt 16080ggccatgggc tgcctggccc gggacttcct gcccagcacc
atttccttca cctggaacta 16140ccagaacaac actgaagtca tccagggtat cagaaccttc
ccaacactga ggacaggggg 16200caagtaccta gccacctcgc aggtgttgct gtctcccaag
agcatccttg aaggttcaga 16260tgaatacctg gtatgcaaaa tccactacgg aggcaaaaac
agagatctgc atgtgcccat 16320tccaggtaag aaccaaaccc tcccagcagg ggtgcccagg
cccaggcatg gcccagaggg 16380agcagcgggt ggggcttagg ccaagctgag ctcacacctt
gacctttcat tccagctgtc 16440gcagagatga accccaatgt gttcgtccca ccacgggatg
gcttctctgg ccctgcacca 16500cgcaagtcta aactcatctg cgaggccacg aacttcactc
caaaaccgat cacagtatcc 16560tggctaaagg atgggaagct cgtggaatct ggcttcacca
cagatccggt gaccatcgag 16620aacaaaggat ccacacccca aacctacaag gtcataagca
cacttaccat ctctgaaatc 16680gactggctga acctgaatgt gtacacctgc cgtgtggatc
acaggggtct caccttcttg 16740aagaacgtgt cctccacatg tgctgccagt gagtggcctg
ggataagccc aatgcctagc 16800cctcccagat tagggaagtc ctcctacaat tatggccaat
gccacccaga catggtcatt 16860tgctccttga actttggctc cccagagtgg ccaaggacaa
gaatgagcaa taggcagtag 16920aggggtgaga atcagctgga aggaccagca tcttccctta
agtaggtttg ggggatggag 16980actaagcttt tttccaactt cacaactaga tatgtcataa
cctgacacag tgttctcttg 17040actgcaggtc cctccacaga catcctaacc ttcaccatcc
ccccctcctt tgccgacatc 17100ttcctcagca agtccgctaa cctgacctgt ctggtctcaa
acctggcaac ctatgaaacc 17160ctgaatatct cctgggcttc tcaaagtggt gaaccactgg
aaaccaaaat taaaatcatg 17220gaaagccatc ccaatggcac cttcagtgct aagggtgtgg
ctagtgtttg tgtggaagac 17280tggaataaca ggaaggaatt tgtgtgtact gtgactcaca
gggatctgcc ttcaccacag 17340aagaaattca tctcaaaacc caatggtagg tatcccccct
tcccttcccc tccaattgca 17400ggacccttcc tgtacctcat agggagggca ggtcctcttc
caccctatcc tcactactgt 17460cttcatttac agaggtgcac aaacatccac ctgctgtgta
cctgctgcca ccagctcgtg 17520agcaactgaa cctgagggag tcagccacag tcacctgcct
ggtgaagggc ttctctcctg 17580cagacatcag tgtgcagtgg cttcagagag ggcaactctt
gccccaagag aagtatgtga 17640ccagtgcccc gatgccagag cctggggccc caggcttcta
ctttacccac agcatcctga 17700ctgtgacaga ggaggaatgg aactccggag agacctatac
ctgtgttgta ggccacgagg 17760ccctgccaca cctggtgacc gagaggaccg tggacaagtc
cactggtaaa cccacactgt 17820acaatgtctc cctgatcatg tctgacacag gcggcacctg
ctattgacca tgctagcgct 17880caaccaggca ggccctgggt gtccagttgc tctgtgtatg
caaactaacc atgtcagagt 17940gagatgttgc attttataaa aattagaaat aaaaaaaatc
cattcaaacg tcactggttt 18000tgattataca atgctcatgc ctgctgagac agttgtgttt
tgcttgctct gcacacaccc 18060tgcatacttg cctccaccct ggcccttcct ctaccttgcc
agtttcctcc ttgtgtgtga 18120actcagtcag gcttacaaca gacagagtat gaacatgcga
ttcctccagc tacttcttga 18180tatatggctg aaagcttgcc taacctggtg caggcagcat
tcaggcacat atatagacac 18240acatgcattt atacatagat atataggtac acatgtgtag
acacatacat gaatgtgtat 18300tcatggacac acagacaaag gtacacatat atacacatga
gttcatgcgc acacacatgc 18360atggacactt acaaacgcct tcagagacaa ataggcatag
acacacaacc actcacagaa 18420acagatacca atatgcatgg tcctgtgtac acagaaacag
actataggca aatatacaca 18480aataaactat atagatacaa agatatgcat atacacacat
gtacagaaac atcttcacat 18540gtgtacacta acatgtggac aggtatagca cacagataca
cctggactct gaccagggct 18600gtaatctcca aggctcacgg ctcagagagc ctacactagg
ctgggtcact gatactcctc 18660aggagcccac tctatgattg ggagagataa ccccaggtac
aaagtatgcc tatctgtctc 18720aacaccatgg ggcagaagat actccactaa ccacccatga
cagaaagtta gccttggctg 18780tgtctccatt aatagaacac ctcagaagac caatgtgaaa
ttgcctaacc cactcacacc 18840caccctgatc tccagttcaa aatgcagaaa acataatgca
gttgtccaaa agatgcccca 18900accacacaca cacacacaca cacacacaca cacacacaca
cacacacaca cacacacaca 18960caccatcaag gagcctctgt aaggagtcac cacccaataa
cactgcctct ttgggctcat 19020atcctggaca ttcttcatat tcatatccat ttggggccta
ggctttagat atccccaagg 19080gctcatcttt acagggatca gagatcccaa taaatgccct
ggtcccacag cctccctcag 19140gtatctgtct gtttatctct tggtaccaag acccaacatt
gctggcaggg gtaggacaag 19200caacgcacgg gaactctgat caaagaaagt catgagatgc
ctgagtcctt caggaagtaa 19260ggagggacaa cctctggtat ccctgttctt attgctaaag
cccaagagac agggagacct 19320gctctaaatt ctcagtctaa acagcaccga tggcaccacc
tgctcaggga aagtccagag 19380cacaccaata tcattttgcc acagttcctg agtctgcctt
tacccaggtc catacattgc 19440atctgtcttg cttgctctgc tgccccaggg ctcctggaac
aaaggctcca aattagtgtg 19500tcctacagct tggcctgttc tgtgcctccg tctagcttga
gctattaggg gaccagtcaa 19560tactcgctaa gattctccag aaccatcagg gcaccccaac
ccttatgcaa atgctcagtc 19620accccaagac ttggcttgac cctccctctc tgtgtccctt
catagagggg gaggtgaatg 19680ctgaggagga aggctttgag aacctgtgga ccactgcctc
caccttcatc gtcctcttcc 19740tcctgagcct cttctacagc accaccgtca ccctgttcaa
ggtagtatgg ttgtggggct 19800gaggacacag ggctgggaca gggagtcacc agtcctcact
gcctctacct ctactcccta 19860caagtggaca gcaattcaca ctgtctctgt cacctgcagg
tgaaatgact ctcagcatgg 19920aaggacagca gagaccaaga gatcctccca cagggacact
acctctgggc ctgggatacc 19980tgactgtatg actagtaaac ttattcttac gtctttcctg
tgttgccctc cagcttttat 20040ctctgagatg gtcttctttc tagactgacc aaagactttt
tgtcaacttg tacaatctga 20100agcaatgtct ggcccacaga cagctgagct gtaaacaaat
gtcacatgga aataaatact 20160ttatcttgtg aactcacttt attgtgaagg aatttgtttt
gtttttcaaa cctttcctgc 20220ggtgttgaca gcccaaggat tatctgaata gagcttagga
actggaaatg gaacagtgca 20280gtctgatggt acttaaggga gaaagaggga aaggaggtgt
ggaagaagaa aaaagagaag 20340cagaggggga ggggagaagg gagagggaga gggagaggga
gagggagagg gagagggaga 20400gggagagaga gagagagaga gagagagaga gagagagcat
gcactctaac agcaaagtac 20460aacacaggca gccaatggat aacactctgg ttatctaccc
tgatggaaga agggaagtag 20520ggcagagaaa attccaggcc taatctccca aaagcaacag
aacctggaaa ctagcctcta 20580gccttaggtc tctgctctgt ccccagccca ccatcttggg
ctggtgttgc ttcaagctag 20640taatttaggt cttatcccaa agctttgtgg tatgtgggtg
tgcctttggg gagttggctg 20700agattttgaa gatgtttgta cctctcccac aacatgacaa
gccctagggg ttagtcaata 20760actcaaattc tctgtctatg acaactgctg tatgactata
tgaagaaatg ggataaagat 20820gctatagtca ctcgaggggg ggcccggtac ccagcttttg
ttccctttag tgagggttaa 20880ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
gtgaaattgt tatccgctca 20940caattccaca caacatacga gccggaagca taaagtgtaa
agcctggggt gcctaatgag 21000tgagctaact cacattaatt gcgttgcgct cactgcccgc
tttccagtcg ggaaacctgt 21060cgtgccagct gcattaatga atcggccaac gcgcggggag
aggcggtttg cgtattgggc 21120gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
cgttcggctg cggcgagcgg 21180tatcagctca ctcaaaggcg gtaatacggt tatccacaga
atcaggggat aacgcaggaa 21240agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
taaaaaggcc gcgttgctgg 21300cgtttttcca taggctccgc ccccctgacg agcatcacaa
aaatcgacgc tcaagtcaga 21360ggtggcgaaa cccgacagga ctataaagat accaggcgtt
tccccctgga agctccctcg 21420tgcgctctcc tgttccgacc ctgccgctta ccggatacct
gtccgccttt ctcccttcgg 21480gaagcgtggc gctttctcat agctcacgct gtaggtatct
cagttcggtg taggtcgttc 21540gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
cgaccgctgc gccttatccg 21600gtaactatcg tcttgagtcc aacccggtaa gacacgactt
atcgccactg gcagcagcca 21660ctggtaacag gattagcaga gcgaggtatg taggcggtgc
tacagagttc ttgaagtggt 21720ggcctaacta cggctacact agaaggacag tatttggtat
ctgcgctctg ctgaagccag 21780ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
acaaaccacc gctggtagcg 21840gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
aaaaggatct caagaagatc 21900ctttgatctt ttctacgggg tctgacgctc agtggaacga
aaactcacgt taagggattt 21960tggtcatgag attatcaaaa aggatcttca cctagatcct
tttaaattaa aaatgaagtt 22020ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
cagttaccaa tgcttaatca 22080gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
catagttgcc tgactccccg 22140tcgtgtagat aactacgata cgggagggct taccatctgg
ccccagtgct gcaatgatac 22200cgcgagaccc acgctcaccg gctccagatt tatcagcaat
aaaccagcca gccggaaggg 22260ccgagcgcag aagtggtcct gcaactttat ccgcctccat
ccagtctatt aattgttgcc 22320gggaagctag agtaagtagt tcgccagtta atagtttgcg
caacgttgtt gccattgcta 22380caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
attcagctcc ggttcccaac 22440gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
agcggttagc tccttcggtc 22500ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
actcatggtt atggcagcac 22560tgcataattc tcttactgtc atgccatccg taagatgctt
ttctgtgact ggtgagtact 22620caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
ttgctcttgc ccggcgtcaa 22680tacgggataa taccgcgcca catagcagaa ctttaaaagt
gctcatcatt ggaaaacgtt 22740cttcggggcg aaaactctca aggatcttac cgctgttgag
atccagttcg atgtaaccca 22800ctcgtgcacc caactgatct tcagcatctt ttactttcac
cagcgtttct gggtgagcaa 22860aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
gacacggaaa tgttgaatac 22920tcatactctt cctttttcaa tattattgaa gcatttatca
gggttattgt ctcatgagcg 22980gatacatatt tgaatgtatt tagaaaaata aacaaatagg
ggttccgcgc acatttcccc 23040gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
cgcggcgggt gtggtggtta 23100cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
cgctcctttc gctttcttcc 23160cttcctttct cgccacgttc gccggctttc cccgtcaagc
tctaaatcgg gggctccctt 23220tagggttccg atttagtgct ttacggcacc tcgaccccaa
aaaacttgat tagggtgatg 23280gttcacgtag tgggccatcg ccctgataga cggtttttcg
ccctttgacg ttggagtcca 23340cgttctttaa tagtggactc ttgttccaaa ctggaacaac
actcaaccct atctcggtct 23400attcttttga tttataaggg attttgccga tttcggccta
ttggttaaaa aatgagctga 23460tttaacaaaa atttaacgcg aattttaaca aaatattaac
gcttacaatt tccattcgcc 23520attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
gcctcttcgc tattacgcca 23580gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
gtaacgccag ggttttccca 23640gtcacgacgt tgtaaaacga cggccagtg
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